Therapeutic and diagnostics proteins comprising a SOCS box

ABSTRACT

The present invention relates generally to therapeutic and diagnostic agents. More particularly, the present invention provides therapeutic molecules capable of modulating signal transduction such as but not limited to cytokine-mediated signal transduction. The molecules of the present invention are useful, therefore, in modulating cellular responsiveness to cytokines as well as other mediators of signal transduction such as endogenous or exogenous molecules, antigens, microbes and microbial products, viruses or components thereof, ions, hormones and parasites.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a continuation-in-part of application Ser. No. 08,962,650, filed on Oct. 31, 1997, a continuation of PCT/AU97/00729 filed Oct. 31, 1997, and also claims priority of U.S. Provisional Application Serial No. 60/083,807, filed on May 1, 1998.

FIELD OF THE INVENTION

The present invention relates generally to therapeutic and diagnostic agents. More particularly, the present invention provides therapeutic molecules capable of modulating signal transduction such as but not limited to cytokine-mediated signal transduction. The molecules of the present invention are useful, therefore, in modulating cellular responsiveness in cytokines as well as other mediators of signal transduction such as endogenous or exogenous molecules, antigens, microbes and microbial products, viruses or components thereof, ions, hormones and parasites.

Bibliographic details of the publications referred to in this specification by author are collected at the end of the description. The subject specification contains nucleotide and amino acid sequence information prepared using the programme PatentIn Version 2.0, presented herein after the bibliography. Each nucleotide or amino acid sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210>1, <210>2, etc).The length, type of sequence (DNA, protein (PRT), etc) and source organism for each nucleotide or amino acid sequence are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide and amino acid sequences referred to in the specification are defined by the information provided in numeric indicator field <400> followed by the sequence identifier (eg. <400>1, <400>2, etc).

The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue. A summary of the sequence listing is given in Table 1.

BACKGROUND OF THE INVENTION

Cells continually monitor their environment in order to modulate physiological and biochemical processes which in turn affects future behaviour. Frequently, a cell's initial interaction with its surroundings occurs via receptors expressed on the plasma membrane. Activation of these receptors, whether through binding endogenous ligands (such as cytokines) or exogenous ligands (such as antigens), triggers a biochemical cascade from the membrane through the cytoplasm to the nucleus.

Of the endogenous ligands, cytokines represent a particularly important and versatile group. Cytokines are proteins which regulate the survival, proliferation, differentiation and function of a variety of cells within the body [Nicole, 1994]. The haemopoietic cytokines have in common a four-alpha helical bundle structure and the vast majority interact with a structurally related family of cell surface receptors, the type I and type II Cytokine receptors. [Bazan, 1990; Sprang, 1993]. In all cases, ligand-induced receptor aggregation appears to be a critical event in initiating intracellular signal transduction cascades. Some cytokines, for example growth hormone, erythropoietin (Epo) and granulocyte-colony-stimulating factor (G-CSF), trigger receptor homodimerisation, while for other cytokines, receptor heterodimerisation or heterotrimerisation is crucial. In the latter cases, several cytokines share common receptor subunits and on this basis can be grouped into three subfamilies with similar patterns of intracellular activation and similar biological effects [Hilton, 1994]. Interleukin-3 (IL-3), IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) use the common β-receptor subunit (βc) and each cytokine stimulates the production and functional activity of granuloctyes and macrophages. IL-2, IL-4, IL-7, IL-9, and IL-15 each use the common γ-chain (γc), while IL-4 and IL-13 share an alternative γ-chain (γ′c or IL-13 receptor α-chain). Each of these cytokines plays an important role in regulating acquired immunity in the lymphoid system. Finally, IL-6, IL-11, leukaemia inhibitory factor (LIF), oncostatin-M (OSM), ciliary neurotrophic factor (CNTF) and cardiotrophin (CT) share the receptor subunit gp 130. Each of these cytokines appears to be highly pleiotropic, having effects both within and outside the haemopoietic system [Nicola, 1994].

In all of the above cases at least one subunit of each receptor complex contains the conserved sequence elements, termed box 1 and box 2, in their ctyoplasmic tails [Murakami, 1991]. Box 1 is a proline-rich motif which is located more proximal to the transmembrane domain than the acidic box 2 element. The box-1 region serves as the binding site for a class of cytoplasmic tyrosine kinases termed JAKs (Janus kinases). Ligand-induced receptor dimerisation serves to increase the catalytic activity of the associated JAKs through cross-phosphorylation. Activated JAKs then tyrosine phosphorylate several substrates, including the receptors themselves. Specific phosphotyrosine residues on the receptor then serve as docking sites for SH2-containing proteins, the best characterised of which are the signal transducers and activators of transcription (STATs) and the adaptor protein, shc. The STATs are then phosphorylated on tyrosines, probably by JAKs, dissociate from the receptor and form either homodimers or heterodimers through the interaction of the SH2 domain of one STAT with the phosphotyrosine residue of the other. STAT dimers then translocate to the nucleus where they bind to specific cytokine-responsive promoters and activate transcription [Darnell, 1994; Ihle, 1995; Ihle, 1995]. In a separate pathway, tyrosine phosphorylated shc interacts with another SH2 domain-containing protein, Grb-2, leading ultimately to activation of members of the MAP kinase family and in turn transcription factors such as fos and jun [Sato, 1993; Cutler, 1993 ]. These pathways are not unique to members of the cytokine receptor family since cytokines that bind receptor tyrosine kinases also being able to activate STATs and members of the MAP kinase family [David, 1996; Lehman, 1996; Shual, 1993; Sato, 199; Cutler, 1993].

Four members of the JAK family of cytoplasmic tyrosine kinases have been described, JAK1, JAK2, JAK3 and TYK2, each of which binds to a specific subset of cytokine receptor subunits. Six STATs have been described (STAT1 through STAT6), and these too are activated by distinct cytokine/receptor complexes. For example, STAT1 appears to be functionally specific to the interferon system, STAT4 appears to be specific to IL-12, while STAT6 appears to be specific for IL-4 and IL-13. Thus, despite common activation mechanisms some degree of cytokine specificity may be achieved through the use of specific JAKs and STATs[Thierfelder, 1996; Kaplan, 1996; Takeda, 1996; Shimoda, 1996; Meraz, 1996; Durbin, 1996].

In addition to those described above, there are clearly other mechanisms of activation of these pathways. For example, the JAK/STAT pathway appears to be able to activate MAP kinases independent of the shc-induced pathway [David, 1995] and the STATs themselves can be activated without binding to the receptor, possibly by direct interaction with JAKs [Gupta, 1996]. Conversely, full activation of STATs may require the action of MAP kinase in addition to that of JAKs [David, 1995; Wen, 1995].

While the activation of these signalling pathways is becoming better understood, little is known of the regulation of these pathways, including employment of negative or positive feedback loops. This is important since once a cell has begun to respond to a stimulus, it is critical that the intensity and duration of the response is regulated and that signal transduction is switched off. It is likewise desirable to increase the intensity of a response systemically or even locally as the situation requires.

In work leading up to the present invention, the inventors sought to isolate negative regulators of signal transduction. The inventors have now identified a new family of proteins which are capable of acting as regulators of signalling. The new family of proteins is defined as the suppressor of cytokine signalling (SOCS) family based on the ability of the initially identified SOCS molecules to suppress cytokine-mediated signalling. It should be noted, however, that not all members of the SOCS family need necessarily share suppressor function nor target solely cytokine mediated signalling. The SOCS family comprises at least three classes of protein molecules based on amino acid sequence motifs located N-terminal of a C-terminal motif called the SOCS box. The identification of this new family of regulatory molecules permits the generation of a range of effector or modulator molecules capable of modulating signal transduction and, hence, cellular responsiveness to a range of molecules including cytokines. The present invention, therefore, provides therapeutic and diagnostic agents based on SOCS proteins, derivatives, homologues, analogues and mimetics thereof as well as agonists and antagonists of SOCS proteins.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

The present invention provides inter alia nucleic acid molecules encoding members of the SOCS family of proteins as well as the proteins themselves. Reference hereinafter to “SOCS” emcompasses any or all members of the SOCS family. Specific SOCS molecules are defined numerically such as, for example, SOCS1, SOCS2 and SOCS3. The species from which the SOCS has been obtained may be indicated by a preface of a single letter abbreviation where “h” is human, “m” is murine and “r” is rat. Accordingly, “mSOCS1” is a specific SOCS from a murine animal. Reference herein to “SOCS” is not to imply that the protein solely suppresses cytokine-mediated signal transduction, as the molecule may modulate other effector-mediated signal transductions such as by hormones or other endogenous or exogenous molecules, antigens, microbes and microbial products, viruses or components thereof, ions, hormones and parasites. The term “modulates” encompasses up-regulation, down-regulation as well as maintenance of particular levels.

One aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a protein or a derivative, homologue, analogue or minetic thereof or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. wherein said protein comprises a SOCS box in its C-terminal region.

Another aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a protein or a derivative, homologue, analogue or mimetic thereof or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. wherein said protein comprises a SOCS box in its C-terminal region and a protein:molecule interacting region.

Yet another aspect of the present invention is directed to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a protein or a derivative, homologue, analogue or mimetic thereof or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. wherein said protein comprises a C-terminal region and a protein:molecule interacting region located in a region N-terminal of the SOCS box.

Preferably, the protein:molecule interacting region is a protein:DNA or protein:protein binding region.

Still a further aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a protein or a derivative, homologue, analogue or mimetic thereof or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. wherein said protein comprises a SOCS box in its C-terminal region and one or more of an SH2domain, WD-40 repeats or ankyrin repeats N-terminal of the SOCS box.

Even still a further aspect of the present invention is directed to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a protein or a derivative, homologue, analogue or mimetic thereof or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. wherein said protein comprises a SOCS box in its C-terminal region wherein the SOCS box comprises the amino acid sequence:

X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ X ₁₂ X ₁₃ X ₁₄ X ₁₅ X ₁₆ X ₁₆ [X _(i]) _(n) X ₁₇ X ₁₈ X ₁₉ X ₂₀ X ₂₁ X ₂₂ X ₂₃ [X _(j]) _(n) x ₂₄ x ₂₅ X ₂₆ X ₂₇ X ₂₈

wherein:

X₁ is L, I, V, M, A or P;

X₂ is any amino acid residue;

X₃ is P, T or S;

X₄ is L, I, V, M, A or P;

X₅ is any amino acid;

X₆ is any amino acid;

X₇ is L, I, V, M, A, F, Y or W;

X₈ is C, T or S;

X₉ is R, K or H;

X₁₀ is any amino acid;

X₁₁ is any amino acid;

X₁₂ is L, I, V, M, A or P;

X₁₃ is any amino acid;

X₁₄ is any amino acid;

X₁₅ is any amino acid;

X₁₆ is L, I, V, M, A, P, G, C, T or S;

[X_(i]) _(n) is a sequence of n amino acids wherein n is from 1 to 50 amino acids and wherein the sequence X_(i) may comprise the same or different amino acids selected from any amino acid residue;

X₁₇ is L, I, V, M, A or P;

X₁₈ is any amino acid;

X₁₉ is any amino acid;

X₂₀ L, I, V, M, A or P;

X₂₁ is P;

X₂₂ is L, I, V, M, A, P or G;

X₂₃ is P or N;

[X_(j]) _(n) is a sequence of n amino acids wherein n is from 0 to 50 amino acids and wherein the sequence X_(j) may comprise the same or different amino acids selected from any amino acid residue;

X₂₄ is L, I, V, M, A or P;

X₂₅ is any amino acid;

X₂₆ is any amino acid;

X₂₇ is Y or F;

X₂₈ is L, I, V, M, A or P;

and a protein:molecule interacting region such as but not limited to one or more of an SH2 domain, WD-40 repeats and/or ankyrin repeats N-terminal of the SOCS box.

Another aspect of the present invention is directed to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a protein or a derivative, homologue, analogue or minetic thereof or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. wherein said protein exhibits the following characteristics:

(i) comprises a SOCS box in its C-terminal region having the amino acid sequence:

X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ X ₁₂ X ₁₃ X ₁₄ X ₁₅ X ₁₆ [X _(i]) _(n) X ₁₇ X ₁₈ X ₁₉ X ₂₀ X ₂₁ X ₂₂ X ₂₃ [X _(j) ] _(n X) ₂₄ X ₂₅ X ₂₆ X ₂₇ X ₂₈

wherein:

X₁ is L, I, V, M, A or P;

X₂ is any amino acid residue;

X₃ is P, T or S;

X₄ is L, I, V, M, A or P;

X₅ is any amino acid;

X₆ is any amino acid;

X₇ is L, I, V, M, A, F, Y or W;

X₈ is C, T or S;

X₉ is R, K or H;

X₁₀ is any amino acid;

X₁₁ is any amino acid;

X₁₂ is L, I, V, M, A or P;

X₁₃ is any amino acid;

X₁₄ is any amino acid;

X₁₅ is any amino acid;

X₁₆ is L, I, V, M, A, P, G, C, T or S;

[X_(i]) _(n) is a sequence of n amino acids wherein n is from 1 to 50 amino acids and wherein the sequence Z_(i) may comprise the same or different amino acids selected from any amino acid residue;

X₁₇ is L, I, V, M, A or P;

X₁₈ is any amino acid;

X₁₉ is any amino acid;

X₂₀ L, I, V, M, A or P;

X₂₁ is P;

X₂₂ is L, I, V, M, A, P or G;

X₂₃ is P or N;

[X_(j]) _(n) is a sequence of n amino acids wherein n is from 0 t 50 amino acids and wherein the sequence X_(j) may comprise the same or different amino acids selected from any amino acid residue;

X₂₄ is L, I, V, M, A or P;

X₂₅ is any amino acid;

X₂₆ is any amino acid;

X₂₇ is Y or F;

X₂₈ is L, I, V, M, A or P; and

(ii) comprises at least one of a SH₂ domain, WD-40 repeats and/or ankyrin repeats or other protein:molecule interacting domain in a region N-terminal of the SOCS box.

Preferably, the SOCS molecules modulate signal transduction such as from a cytokine or hormone or other endogenous or exogenous molecule, a microbe or microbial product, an antigen or a parasite.

More preferably, the SOCS molecule modulate cytokine mediated signal transduction.

Still another aspect of the present invention comprises a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a protein or a derivative, homologue, analogue or mimetic thereof or comprises a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. wherein said protein exhibits the following characteristics;

(i) is capable of modulating signal transduction;

(ii) comprises a SOCS box in its C-terminal region having the amino acid sequence:

X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ X ₁₂ X ₁₃ X ₁₄ X ₁₅ X ₁₆ [X _(i]) _(n) X ₁₇ X ₁₈ X ₁₉ X ₂₀ X ₂₁ X ₂₂ X ₂₃ [X _(j]) _(n) X ₂₄ X ₂₅ X ₂₆ X ₂₇ X ₂₈

wherein:

X₁ is L, I, V, M, A or P;

X₂ is any amino acid residue;

X₃ is P, T or S;

X₄ is L, I, V, M, A or P;

X₅ is any amino acid;

X₆ is any amino acid;

X₇ is L, I, V, M, A, F, Y or W;

X₈ is C, T or S;

X₉ is R, K or H;

X₁₀ is any amino acid;

X₁₁ is any amino acid;

X₁₂ is L, I, V, M, A or P;

X₁₃ is any amino acid;

X₁₄ is any amino acid;

X₁₅ is any amino acid;

X₁₆ is L, I, V, M, A, P, G, C, T or S;

[X_(i]) _(n) is a sequence of n amino acids wherein n is from 1 to 50 amino acids and wherein the sequence X_(i) may comprise the same or different amino acids selected from any amino acid residue;

X₁₇ is L, I, V, M, A or P;

X₁₈ is any amino acid;

X₁₉ is any amino acid;

X₂₀ L, I, V, M, A or P;

X₂₁ is P;

X₂₂ is L, I, V, M, A, P or G;

X₂₃ is P or N;

[X_(j]) _(n) is a sequence of n amino acids wherein n is from 0 to 50 amino acids and wherein the sequence X_(j) may comprise the same or different amino acids selected from any amino acid residue;

X₂₄ is L, I, V, M, A or P;

X₂₅ is any amino acid;

X₂₆ is any amino acid;

X₂₇ is Y or F;

X₂₈ is L, I, V, M, A or P; and

(iii) comprises at least one of a SH2 domain, WD-40 repeats and/or ankyrin repeats or other protein:molecule interacting domain in a region N-terminal of the SOCS box.

Preferably, the signal transduction is mediated by a cytokine such as one or more of EPO, TPO, G-CSF, GM-CSF, IL-3, IL-4, IL-7, IL-13, IL-6, LIF, IL-12, IFNα, TNFα, IL-1 and/or M-CSF.

Preferably, the signal transduction is mediated by one of more of Interleukin 6 (IL-6), Leukaemia Inhibitory Factor (LIF), Oncostatin M (OSM), Interferon (IFN)-α and/or thrombopoietin.

Preferably, the signal transduction is mediated by IL-6.

Particularly preferred nucleic acid molecules comprise nucleotide sequences substantially set forth in SEQ ID No: 3 (mSOCS1), SEQ ID NO: 5 (mSOCS2), SEQ ID NO: 7 (mSOCS3), SEQ ID NO: 9 (hSOCS1), SEQ ID NO: 11 (rSOCS1), SEQ ID NO: 13 (mSOCS4), SEQ ID NOS: 15 and 16 (hSOCS4), SEQ ID NO: 17 (mSOCS5), SEQ ID NO: 19 (h,SOCS5), SEQ ID NO: 20 (mSOCS6), SEQ ID NOS: 22 and 23 (hSOCS6), SEQ ID NO: 24 (mSOCS7),SEQ ID NOS: 26 and 27 (hSOCS7), SEQ ID NO: 28 (mSOCS8), SEQ ID NO: 30 (mSOCS9), SEQ ID NO: 31 (hSOCS9), SEQ ID NO: 32 (mSOCS10), SEQ ID NOS: 33 and 34 (hSOCS10), SEQ ID NO: 35 (hSOCS11), SEQ ID NO: 37 (mSOCS12), SEQ ID NOS: 38 and 39 (hSOCS12), SEQ ID NO: 40 (mSOCS13), SEQ ID NO: 42 (hSOCs13), SEQ ID NO: 43 (mSOCS14), SEQ ID NO: 45 (mSOCS15) and SEQ ID NO: 47 (hSOCS15) or a nucleotide sequence having at least about 15% similarity to all or a region of any of the listed sequences or a nucleotide acid molecule capable of hybridizing to any one of the listed sequences under low stringency conditions at 42° C.

Another aspect of the present invention relates to a protein or a derivative, homologue, analogue or mimetic thereof comprising a SOCS box in its C-terminal region.

Yet another aspect of the present invention is directed to a protein or a derivative, homologue, analogue or minetic thereof comprising a SOCS box in its C-terminal region and a protein:molecule interacting region.

Even yet another aspect of the present invention provides a protein or a derivative, homologue, analogue or minetic thereof comprising an interacting region located in a region N-terminal of the SOCS box.

Preferably, the protein:molecule interacting region is a protein:DNA or a protein:protein binding region.

Another aspect of the present invention contemplates a protein or a derivative, homologue, analogue or mimetic thereof comprising a SOCS box in its C-terminal region and a SH₂ domain, WD-40 repeats or ankyrin repeats N-terminal of the SOCS box.

Still yet another aspect of the present invention provides a protein or a derivative, homologue, analogue or minetic thereof exhibiting the following characteristics:

(i) comprises a SOCS box in its C-terminal region having the amino acid sequence:

X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ X ₁₂ X ₁₃ X ₁₄ X ₁₅ X ₁₆ [X _(i]) _(n) X ₁₇ X ₁₈ X ₁₉ X ₂₀ X ₂₁ X ₂₂ X ₂₃ X ₂₄ X ₂₅ X ₂₆ X ₂₇ C ₂₈

wherein:

X₁ is L, I, V, M, A or P;

X₂ is any amino acid residue;

X₃ is P, T or S;

X₄ is L, I, V, M, A or P;

X₅ is any amino acid;

X₆ is any amino acid;

X₇ is L, I, V, M, A, F, Y or W;

X₈ is C, T or S;

X₉ is R, K or H;

X₁₀ is any amino acid;

X₁₁ is any amino acid;

X₁₂ is L, I, V, M, A or P;

X₁₃ is any amino acid;

X₁₄ is any amino acid;

X₁₅ is any amino acid;

X₁₆ is L, I, V, M, A, P, G, C, T or S;

[X_(i]) _(n) is a sequence of n amino acids wherein n is from 1 to 50 amino acids and wherein the sequence X_(i) may comprise the same or different amino acids selected from any amino acid residue;

X₁₇ is L, I, V, M, A or P;

X₁₈ is any amino acid;

X₁₉ is any amino acid;

X₂₀ L, I, V, M, A or P;

X₂₁ is P;

X₂₂ is L, I, V, M, A, P or G;

X₂₃ is P or N;

[_(j]) _(n) is a sequence of n amino acids wherein n is from 0 to 50 amino acids and wherein the sequence X_(j) may comprise the same or different amino acids selected from any amino acid residue;

X₂₄ is L, I, V, M, A or P;

X₂₅ is any amino acid;

X₂₆ is any amino acid;

X₂₇ is Y or F;

X₂₈ is L, I, V, M, A or P; and

(ii) comprises at least one of a SH2 domain, WD-40 repeats and/or ankyrin repeats or other protein:molecule interacting domain in a region N-terminal of the SOCS box.

Preferably, the proteins modulate signal transduction such as cytokine-mediated signal transduction.

Preferred cytokines are EPO, TPO, G-CSF, GM-CSF, IL-3, IL-2, IL-4, IL-7, IL-13, IL-6, LIF, IL-12, IFNγ, TNFα, IL-1 and/or M-CSF.

A particularly preferred cytokineis IL-6.

Even yet another aspect of the present invention provides a protein or derivative, homologue, analogue or minetic thereof exhibiting the following characteristics:

(i) is capable of modulating signal transduction such as cytokine-mediated signal transduction;

(ii) comprises a SOCS box in its C-terminal region having the amino acid sequence:

X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ X ₁₂ X ₁₃ X ₁₄ X ₁₅ X ₁₆ [X _(i]) _(n) X ₁₇ X ₁₈ X ₁₉ X ₂₀ X ₂₁ X ₂₂ X ₂₃ [X _(j]) _(n) X ₂₄ X ₂₅ X ₂₆ X ₂₇ X ₂₈

wherein:

X₁ is L, I, V, M, A or P;

X₂ is any amino acid residue;

X₃ is P, T or S;

X₄ is L, I, V, M, A or P;

X₅ is any amino acid;

X₆ is any amino acid;

X₇ is L, I, V, M, A, F, Y or W;

X₈ is C, T or S;

X₉ is R, K or H;

X₁₀ is any amino acid;

X₁₁ is any amino acid;

X₁₂ is L, I, V, M, A or P;

X₁₃ is any amino acid;

X₁₄ is any amino acid;

X₁₅ is any amino acid;

X₁₆ is L, I, V, M, A, P, G, C, T or S;

[X_(j]) _(n) is a sequence of n amino acids wherein n is from 1 to 50 amino acids and wherein the sequence X_(i) may comprise the same or different amino acids selected from any amino acid residue;

X₁₇ is L, I, V, M, A or P;

X₁₈ is any amino acid;

X₁₉ is any amino acid;

X₂₀ L, I, V, M, A or P;

X₂₁ is P;

X₂₂ is L, I, V, M, A, P or G;

X₂₃ is P or N;

_(n) is a sequence of n amino acids wherein n is from 0 to 50 amino acids and wherein the sequence X_(j) may comprise the same or different amino acids selected from any amino acid residue;

X₂₄ is L, I, V, M, A or P;

X₂₅ is any amino acid;

X₂₆ is any amino acid;

X₂₇ is Y or F;

X₂₈ is L, I, V, M, A or P; and

(iii) comprises at least one of a SH2 domain, WD-40 repeats and/or ankyrin repeats or other protein-molecule interacting domain in a region N-terminal of the SOCS box.

Particularly preferred SOCS proteins comprise an amino acid sequence substantially as set forth in SEQ ID NO: 4 (mSOCS1), SEQ ID NO: 6 (mSOCS2), SEQ ID NO: 8 (mSOCS3), SEQ ID NO: 10 (hSOCS1), SEQ ID NO: 12 (rSOCS1), SEQ ID NO: 14 (mSOCS4), SEQ ID NO: 18 (mSOCS5), SEQ ID NO: 21 (mSOCS6), SEQ ID NO: 25 (mSOCS7), SEQ ID NO: 29 (mSOCS8), SEQ ID NO: 36 (hSOCS11), SEQ ID NO: 41 (mSOCS13), SEQ ID NO: 44 (mSOCS14), SEQ ID NO: 46 (mSOCS15) and SEQ ID NO: 48 (hSOCS15) or an amino acid sequence having at least 15% similarity to all or a region of any one of the listed sequences.

Another aspect of the present invention contemplates a method of modulating levels of a SOCS protein in a cell said method comprising contacting a cell containing a SOCS gene with an effective amount of a modulator of SOCS gene expression or SOCS protein activity for a time and under conditions sufficient to modulate levels of said SOCS protein.

A related aspect of the present invention provides a method of modulating signal transduction in a cell containing a SOCS gene comprising contacting said cell with an effective amount of a modulator of SOCS gene expression or SOCS protein activity for a time sufficient to modulate signal transduction.

Yet a further related aspect of the present invention is directed to a method of influencing interaction between cells wherein at least one cell carries a SOCS gene, said method comprising contacting the cell carrying the SOCS gene with an effective amount of a modulator of SOCS gene expression or SOCS protein activity for a time sufficient to modulate signal transduction.

In accordance with the present invention, n in [X_(i)]_(n) and [X_(j)]_(n) may, in addition from being 1-50, be from 1-30, 1-20, 1-10 and 1-5.

A summary of the sequence listing referred to in the subject specification is given in Table 1.

TABLE 1 SUMMARY OF SEQUENCE IDENTIFYING NUMBERS SEQUENCE SEQ ID NO PCR Primer 1 PCR Primer 2 Mouse SOCS1 (nucleotide) 3 Mouse SOCS1 (amino acid) 4 Mouse SOCS2 (nucleotide) 5 Mouse SOCS2 (amino acid) 6 Mouse SOCS3 (nucleotide) 7 Mouse SOCS3 (amino acid) 8 Human SOCS1 (nucleotide) 9 Human SOCS1 (amino acid) 10 Rat SOCS1 (nucleotide) 11 Rat SOCS1 (amino acid) 12 nucleotide sequence of murine SOCS4 13 amino acid sequence of murine SOCS4 14 nucleotide sequence of SOCS4 cDNA human contig 4.1 15 nucleotide sequence of SOCS4 cDNA human contig 4.2 16 nucleotide sequence of murine SOCS5 17 amino acid sequence of murine SOCS5 18 nucleotide sequence of human SOCS5 19 nucleotide sequence of murine SOCS6 20 amino acid of murine SOCS6 21 nucleotide sequence of human SOCS6 contig h6.1 22 nucleotide sequence of human SOCS6 contig h6.2 23 nucleotide sequence of murine SOCS7 24 amino acid sequence of murine SOCS7 25 nucleotide sequence of human SOCS7 contig h7.1 26 nucleotide sequence of human SOCS7 contig 17.2 27 nucleotide sequence of murine SOCS8 28 amino acid sequence of murine SOCS 8 29 nucleotide sequence of murine SOCS9 30 nucleotide sequence of human SOCS9 31 nucleotide sequence of murine SOCS10 32 nucleotide sequence of human SOCS10 contig h10.1 33 nucleotide sequence of human SOCS10 contig h10.2 34 nucleotide sequence of human SOCS11 35 amino acid sequence of human SOCS11 36 nucleotide sequence of mouse SOCS12 37 nucleotide sequence of human SOCS12 contig h12.1 38 nucleotide sequence of human SOCS12 contig h12.2 39 nucleotide sequence of murine SOCS13 40 amino acid sequence of murine SOCS13 41 nucleotide sequence of human SOCS13 cDNA contig h13.1 42 nucleotide sequence of murine SOCS14 cDNA 43 amino acid sequence of murine SOCS14 44 nucleotide sequence of murine SOCS15 cDNA 45 amino acid sequence of murine SOCS15 46 nucleotide sequence of human SOCS15 47 amino acid sequence of human SOCS15 48 5′ oligonucleotide sequence (2465) 49 3′ oligonucleotide sequence (2466) 50 N-terminal GFP tag 51 3′ genomic oligonucleotide no. 3243 52 5′ genomic oligonucleotide no. 3244 53 Amino acid sequence of SEQ ID NO: 53 54 3′ cDNA oligonucleotide no. 3245 55 Nucleotide sequence of murine SOCS13 56 Amino acid sequence of murine SOCS13 57 Oligonucleotide no. 3342 58 Amino acid sequence of SOCS box 59 Nucleotide sequence of murine SOCS5 60 Amino acid sequence of murine SOCS5 61 Nucleotide sequence of murine SOCS9 62 Amino acid sequence of murine SOCS9 63

Single and three letter abbreviations are used to denote amino acid residues and these are summarized in Table 2.

TABLE 2 Three-letter One-letter Amino Acid Abbreviation Symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Any residue Xaa X

BRIEF DESCRIPTION OF THE DRAWINGS

In some of the Figures, abbreviations are used to denote SOCS proteins with certain binding motifs. SOCS proteins which contain WD-40 repeats are referred to as WSB-1-WSB4. SOCS proteins with ankyrin repeats are referred to as ASB1-ASB3. Deletion mutants are “Δ”. For example, ΔC is a deletion in the carboxy terminal region and ΔN is a deletion in the amino terminal region.

FIG. 1 is a diagrammatic representation showing generation of an IL-6-unresponsive M1 clone by retroviral infection. The RUFneo retrovirus, showing the position of landmark restriction endonuclease cleavage sites, the 4A2 cDNA insert and the position of PCR primer sequences.

FIGS. 2A-2B depicts the nucleotide sequence and structure of the SOCS1 gene. 2A. The genomic context of SOCS1 in relation to the protamine gene cluster on murine chromosome 16. The accession number of the locus is MMPRMGNS (direct submission; G. Schlueter, 1995) for the mouse and BTPRMTNP2 for the rat (direct submission; G. Schlueter, 1996). 2B. The nucleotide sequence of the SOCS1 cDNA and deduced amino acid sequence. Conventional three letter abbreviations are used for the amino acid sequence and the asterisk indicates the stop codon. The polyadenylation signal sequence is underlined. The coding region is shown in uppercase and the untranslated region is shown in lower case.

FIG. 3 is a representation of a comparison of the amino acid sequences of SOCS1, SOCS2, SOCS3 and CIS. Alignment of the predicted amino acid sequence of mouse (mm), human (hs) and rat (rr) SOCS1, SOCS2, SOCS3 and CIS. Those residues shaded are conserved in three or four mouse SOCS family members. The SH2 domain is boxed in dotted lines, while the SOCS box is bounded by double lines.

FIGS. 4A(I)-4F(II) show a comparison of the amino acid sequence of the SOCS proteins. Schematic representation of structures of SOCS proteins including proteins which contain WD-40 repeats (WSB) and ankyrin repeats (ASB). 4A(I)-4A(II): Alignment of N-terminal regions of SOCS proteins. 4B(I)-4B(II): Alignment of the SH2 domains of CIS, SOCS1, 2, 3, 5, 9, 11 and 14. 4C(I)-4C(III): Alignment of the WD-40 repeats of SOCS4, SOCS6, SOCS13 and SOCS15. 4D: Alignment of the ankyrin repeats of SOCS7 and SOCS10. 4E(I)-4E(II): Alignment of the regions between SH2, WD-40 and ankyrin repeats and the SOCS box. 4F(I)-4F(II): Alignment of the SOCS box. In each case the conventional one letter abbreviations for amino acids are used, with X denoting residues of uncertain identity and ◯◯◯ denoting the beginning and the end of contigs. Amino acid sequence obtained from conceptual translation of nucleic acid sequence derived from isolated cDNAs is shown in upper case while amino acid sequence obtained by conceptual translation of ESTs is shown in lower case and is approximate only. Conserved residues, defined as (LIVMA), (FYW), (DE), (QN), (C, S, T), (KRH), (PG) are shaded in the SH2 domain, WD-40 repeats, ankyrin repeats and the SOCS box. For the alignment of SH2 domains, WD-40 repeats and ankyrin repeats a consensus sequence is shown above. In each case this has been derived from examination of a large and diverse set of domains (Neer et al, 1994; Bork, 1993).

FIG. 5 is a representation showing the nucleotide sequence of the mouse SOCS4 cDNA. The nucleotides encoding the mature coding region from the predicted ATG “start” codon to the stop codon is shown in upper case, while the predicted 5′ and 3′ untranslated regions are shown in lower case.

FIG. 6 is a representation showing the predicted amino acid sequence of the mouse SOCS4 protein, derived from the nucleotide sequence in FIG. 5. The SOCS box, which also shown in FIG. 4, is underlined.

FIG. 7 is a representation showing the nucleotide sequence of human SOCS4 cDNA contigs h4.1 and h4.2, derived from analysis of ESTs listed in Table 4.1.

FIG. 8 is a diagrammatic representation showing the relationship of mouse SOCS5 genomic (57-2) and cDNA (5-3-2) clones to contigs derived from analysis of mouse ESTs (Table 5.1) and human cDNA clone (5-94-2) and ESTs (Table 5.2). The nucleotide sequence of the mouse SOCS5 contig is shown in FIG. 9A, with the sequence of human SOCS5 contig (h5.1) being shown in FIG. 10. The deduced amino acid sequence of mouse SOCS5 is shown in FIG. 9B. The structure of the protein is shown schematically, with the SH2 domain indicated by the open white wave box and the SOCS box by the open hatched box. The putative 5′ and 3′ translated regions are shown by the thin solid line.

FIG. 9A is a representation showing the nucleotide sequence of the mouse SOCS5 derived from analysis of genomic and cDNA clones. The nucleotides encoding the mature coding region from the predicted ATG “start” codon to the stop codon is shown in upper case, while the predicted 5′ and 3′ untranslated regions are shown in lower case. The relationship of mouse cDNA sequence to mouse and human EST contigs is illustrated in FIG. 8.

FIG. 9B is a representation of the predicted amino acid sequence of mouse SOCS5 protein, derived from the nucleotide sequence in FIG. 9A. The SOCS box, which also shown in FIG. 4 is underlined.

FIG. 10 is a representation showing the nucleotide sequence of human SOCS5 cDNA contig h5.1, derived from analysis of cDNA clone 5-94-2 and the ESTs listed in Table 5.2. The relationship of these contigs to the mouse cDNA sequence is illustrated in FIG. 8.

FIG. 11 is a diagrammatic representation showing the relationship of mouse SOCS6 cDNA clones (6-1A, 6-2A, 6-5B, 6-4N, 6-18, 6-29, 6-3N and 6-5N) to contigs derived from analysis of mouse ESTs (Table 6.1) and human ESTs (Table 6.2). The nucleotide sequence of the mouse SOCS-6 contig is shown in FIG. 12A, with the sequence of human SOCS6 contigs (h6.1 and h6.2) being shown in FIG. 13. The deduced amino acid sequence of mouse SOCS6 is shown in FIG. 12B. The structure of the protein is shown schematically, while the WD-40 repeats indicated by the open hatched boxes and the SOCS box by the open dotted box. The putative 5′ and 3′ untranslated regions are shown by the thin solid line.

FIG. 12A is a representation showing the nucleotide sequence of the mouse SOCS6 derived from analysis of cDNA clone 64-10A-11. The nucleotides encoding the part of the predicted coding region, ending in the stop codon are shown in upper case, while the predicted 3′ untranslated regions are shown in lower case. The relationship of mouse cDNA sequence to mouse and human EST contigs is illustrated in FIG. 11.

FIG. 12B is a representation showing the predicted amino acid sequence of mouse SOCS6 protein, derived from the nucleotide sequence in FIG. 12A. The SOCS box, which also shown in FIG. 4 is underlined.

FIG. 13 is a representation showing the nucleotide sequence of human SOCS6 cDNA contig h6.1, and contig h6.2, derived from analysis of cDNA clone 5-94-2 and the ESTs listed in Table 6.2. The relationship of these contigs to the mouse cDNA sequence is illustrated in FIG. 11.

FIG. 14 is a diagrammatic representation showing the relationship of mouse SOCS7 cDNA clone (74-10A-11) to contigs derived from analysis of mouse ESTs (Table 7.1) and human ESTs (Table 7.2). The nucleotide sequence of the mouse SOCS7 contig is shown in FIG. 15A with the sequence of human SOCS7 contigs (h7.1 and h7.2) being shown in FIG. 16. The deduced amino acid sequence of mouse SOCS7 is shown in FIG. 15B. The structure of the protein is shown schematically, with the ankyrin repeats indicated by the cross hatched box and the SOCS box by the open dotted box. The putative 5′ and 3′ untranslated regions are shown by the thin solid line in the mouse and by the wavy line in h7.2. Based on analysis of clones isolated to date and ESTs the 3′ untranslated regions of mSOCS7 and hSOCS7 share little similarity.

FIG. 15A is a representation showing the nucleotide sequence of the mouse SOCS7 derived from analysis of cDNA clone 74-10A-11. The nucleotides encoding the part of the predicted coding region, ending in the stop codon are shown in upper case, while the predicted 3′ untranslated regions are shown in lower case. The relationship of mouse cDNA sequence to mouse and human EST contigs is illustrated in FIG. 14.

FIG. 15B is a representation showing the predicted amino acid sequence of mouse SOCS7 protein, derived from the nucleotide sequence in FIG. 15A. The SOCS box, which also shown in FIG. 4 is underlined.

FIG. 16 is a representation showing the nucleotide sequence of human SOCS7 cDNA contig h7.1 and h7.2 derived from analysis of the ESTs listed in Table 7.2. The relationship of these contigs to the mouse cDNA sequence is illustrated in FIG. 14.

FIG. 17 is a diagrammatic representation of the relationship of sequence derived from analysis of mouse SOCS8 ESTs (Table 8.1) to the predicted protein structure of mouse SOCS8. The deduced partial amino acid sequence of mouse SOCS8 is shown in FIG. 18B. The structure of the protein is shown schematically with the SOCS box indicated by the cross hatched box. The predicted 3′ untranslated region is shown by the thin line.

FIG. 18A is a representation showing the partial nucleotide sequence of mouse SOCS8 cDNA (contig 8.1) derived from analysis of ESTs. The nucleotides encoding the part of the predicted coding region, ending in the STOP codon are shown in upper case, while the predicted 3′ untranslated regions are shown in lower case.

FIG. 18B is a representation showing the partial predicted amino acid sequence of the mouse SOCS8 protein, derived from the nucleotide sequence in FIG. 18A. The SOCS box, which also shown in FIG. 4 is underlined.

FIG. 19 is a diagrammatic representation showing the relationship of mouse SOCS9 ESTs (Table 9.1) and human SOCS9 ESTs (Table 9.2). The nucleotide sequence of the mouse SOCS9 contig (m9.1) is shown in FIG. 20, with the sequence of human SOCS9 contig (h9.1) being shown in FIG. 21. The deduced amino acid sequence of human SOCS9 is shown schematically, with the SH2 domain indicated by the open white wave box and the SOCS box by the open hatched box. The putative 3′ untranslated region is shown by the thin solid line.

FIG. 20 is a representation showing the partial nucleotide sequence of mouse SOCS9 cDNA (contig m9.1), derived from analysis of the ESTs listed in Table 9.1. The relationship of these contigs to the mouse cDNA sequence is illustrated in FIG. 19.

FIG. 21 is a representation showing the partial nucleotide sequence of human SOCS9 cDNA (contig h9.1), derived from analysis of the ESTs listed in Table 9.2. Although it is clear that contig h9.1 encodes a protein with an SH2 domain and a SOCS box, the quality of the sequence is not high enough to derive a single unambiguous open reading frame. The relationship of these contigs to the mouse cDNA sequence is illustrated in FIG. 19.

FIG. 22 is a representation showing the relationship of mouse SOCS10 cDNA clones (10-9, 10-12, 10-23 and 10-24) to contigs derived from analysis of mouse ESTs (Table 10.1) and human ESTs (Table 10.2). The nucleotide sequence of the mouse SOCS10 contig is shown in FIG. 23, with the sequence of human SOCS10 contigs (h10.1 and h10.2) being shown in FIG. 24. The predicted structure of the protein is shown schematically, with the ankyrin repeats indicated by the cross hatched box and the SOCS box by open hatched box. The putative 3′ untranslated regions is shown by the thin line solid line in the mouse and by the wavy line in h10.2. Based on analysis of clones isolated to date and ESTs the 3′ untranslated regions of mSOCS-10 and hSOCS-10 share little similarity.

FIG. 23 is a representation showing the nucleotide sequence of the mouse SOCS10 derived from analysis of cDNA clone 10-9, 10-12, 10-23 and 10-24. The nucleotides encoding the part of the predicted coding region, ending in the stop codon are shown in upper case, while the predicted 3′ untranslated regions are shown in lower case. Although it is clear that contig m10.1 encodes a protein with a series of ankyrin repeats and a SOCS box, the quality of the sequence is not high enough to derive a single unambiguous open reading frame. The relationship of mouse cDNA sequence to mouse and human EST contigs is illustrated in FIG. 22.

FIG. 24 is a representation showing the nucleotide sequence of human SOCS10 cDNA contig h10.2 and h10.2 derived from analysis of the ESTs listed in Table 10.2. The relationship of these contigs to the mouse cDNA sequence is illustrated in FIG. 22.

FIG. 25A is a representation showing the partial nucleotide sequence of the human SOCS11 cDNA derived from analysis of ESTs listed in Table 11.1 The nucleotides encoding the mature coding region from the predicted ATG “start” codon to the stop codon is shown in upper case, while the predicted 5′ and 3′ untranslated regions are shown in lower case. The relationship of the partial cDNA sequence, derived from ESTs, to the predicted protein is shown in FIG. 26.

FIG. 25B is a representation showing the partial predicted amino acid sequence of human SOCS11 protein, derived from the nucleotide sequence in FIG. 25A. The SOCS box, which also shown in FIG. 4, is underlined.

FIG. 26 is a diagrammatic representation showing the relationship of sequence derived from analysis of human SOCS-11 ESTs (Table 11.1) to the predicted protein structure of human SOCS11. The deduced partial amino acid sequence of human SOCS11 is shown in FIG. 25B. The structure of the protein is shown schematically with the SH2 domain shown by the open hatched box and the SOCS box shown by the open white box. The predicted 3′ untranslated region is shown by the thin line.

FIG. 27 is a diagrammatic representation showing the relationship of mouse SOCS12 cDNA clones (12-1) to contigs derived from analysis of mouse ESTs (Table 12.1) and human ESTs (Table 12.2). The nucleotide sequence of the mouse SOCS12 contig is shown in FIG. 28, with the sequence of human SOCS12 contigs (h12.1 and h12.2) being shown in FIG. 29. The structure of the protein is shown schematically, with the ankyrin repeats indicated by the cross hatched box and the SOCS box by the open hatched box. The putative 3′ untranslated region is shown by the thin line solid line in the mouse and by the wavy line in h12.2. Based on analysis of clones isolated to date and ESTs the 3′ untranslated regions of mSOCS12 and hSOCS12 share little similarity.

FIG. 28 is a representation showing the nucleotide sequence of the mouse SOCS12 derived from analysis of cDNA clone 12-1 and the ESTs listed in Table 12.1. The nucleotides encoding the part of the predicted coding region, including the stop codon are shown in upper case, while the predicted 3′ untranslated region is shown in lower case. By homology with human SOCS12 it is clear that contig m12.1 encodes a protein with a series of ankyrin repeats and a SOCS box, the quality of the sequence is not high enough to derive a single unambiguous open reading frame. The relationship of mouse cDNA sequence to mouse and human EST contigs is illustrated in FIG. 27.

FIG. 29 is a representation showing the nucleotide sequence of human SOCS12 cDNA contig h12.1 and h12.2 derived from analysis of the ESTs listed in Table 12.2. The relationship of these contigs to the mouse cDNA sequence is illustrated in FIG. 27.

FIG. 30 is a diagrammatic representation showing the relationship of contig m13.1 derived from analysis of mouse SOCS13 cDNA clones (62-1, 62-6-7, 62-14) and mouse ESTs (Table 13.1) to contig h13.1 derived from analysis of human ESTs (Table 13.2). The nucleotide sequence of the mouse SOCS13 contig is shown in FIG. 31A, with the sequence of human SOCS13 contig (h13.1) being shown in FIG. 32. The deduced amino acid sequence of mouse SOCS13 is shown in FIG. 31B. The structure of the protein is shown schematically, with the WD-40 repeats highlighted by the open hatched box and the SOCS box shown by the densely hatched box. The 3′ untranslated region is shown by the thin line solid line.

FIG. 31A is a representation showing the nucleotide sequence of the mouse SOCS13 derived from analysis of cDNA clone 62-1, 62-6-7 and 62-14. The nucleotides encoding part of the predicted coding region, ending in the stop codon are shown in upper case, while those encoding the predicted 3′ untranslated regions are shown in lower case. The relationship of mouse cDNA sequence to mouse and human EST contigs is illustrated in FIG. 30.

FIG. 31B is a representation showing the predicted amino acid sequence of mouse SOCS13 protein, derived from the nucleotide sequence in FIG. 31A. The SOCS box, which also shown in FIG. 4 is underlined.

FIG. 32 is a representation showing the nucleotide sequence of human SOCS13 cDNA contig h13.1 derived from analysis of the ESTs listed in Table 13.2. The relationship of these contigs to the mouse cDNA sequence is illustrated in FIG. 30.

FIG. 33 is a diagrammatic representation showing the relationship of a partial mouse SOCS14 cDNA clone (14-1) to contigs derived from analysis of mouse ESTs (Table 14.1). The nucleotide sequence of the mouse SOCS14 contig is shown in FIG. 34A. The deduced partial amino acid sequence of mouse SOCS14 is shown in FIG. 34B. The structure of the protein is shown schematically, with the SH2 domain indicated by the open wave box and the SOCS box by the densely hatched box. The putative 3′ untranslated region is shown by the thin line.

FIG. 34A is a representation showing the nucleotide sequence of the mouse SOCS14 derived from analysis of genomic and cDNA clones. The nucleotides encoding the mature coding region from the predicted ATG “start” codon to the stop codon is shown in upper case, while the predicted 5′ and 3′ untranslated regions are shown in lower case. The relationship of mouse cDNA sequence to mouse and human EST contigs is illustrated in FIG. 33.

FIG. 34B is a representation showing the predicted amino acid sequence of mouse SOCS14 protein, derived from the nucleotide sequence in FIG. 34A. The SOCS box, which also shown in FIG. 4 is underlined.

FIG. 35 is a diagrammatic representation showing the relationship of contig m15.1 derived from analysis of mouse BAC and mouse ESTs (Table 15.1) to contig h15.1 derived from analysis of the human BAC and human ESTs (Table 15.2). The nucleotide sequence of the mouse SOCS15 contig is shown in FIG. 36A, with the sequence of human SOCS15 contig (h15.1) being shown in FIG. 38A. The deduced amino acid sequence of mouse SOCS15 is shown in FIG. 36B. The structure of the protein is shown schematically, with the WD-40 repeats highlighted by the open hatched box on the left and the SOCS box highlighted by the hatched box on the right. The 5′ and 3′ untranslated region are shown by the thin line solid line. The introns which interrupt the coding region are shown by Λ.

FIGS. 36A(i)-(iii) is a representation showing the nucleotide sequence covering the mouse SOCS15 gene derived from analysis the mouse BAC listed in Table 15.1. The nucleotides encoding the predicted coding region, beginning with the ATG and ending in the stop codon are shown in upper case, while those encoding the predicted 5′ untranslated region, the introns and the 3′ untranslated region are shown in lower case. The relationship of mouse BAC to mouse and human ESTs contigs is illustrated in FIG. 35.

FIG. 36B is a representation showing the predicted amino acid sequence of mouse SOCS15 protein, derived from the nucleotide sequence in FIG. 36A. The SOCS box, which also shown in FIG. 4 is underlined.

FIGS. 37A(i)-(iv) is a representation showing the nucleotide sequence covering the human SOCS15 gene derived from analysis the human BAC listed in Table 15.2. The nucleotides encoding the predicted coding region, beginning with the ATG and ending in the stop codon are shown in upper case, while those encoding the predicted 5′ untranslated region, the introns and the 3′ untranslated region are shown in lower case. The relationship of the human BAC to mouse and human ESTs contigs is illustrated in FIG. 35.

FIG. 37B is a representation showing the predicted amino acid sequence of human SOCS15 protein, derived from the nucleotide sequence in FIG. 37A. The SOCS box, which also shown in FIG. 4 is underlined.

FIG. 38 is a diagrammatic representation of pβgalpAloxneo.

FIG. 39 is a diagrammatic representation of pβgalpAloxneoTK.

FIG. 40 is a diagrammatic representation of SOCS1 knockout construct.

FIGS. 41A-C is the nucleotide sequence and predicted amino acid sequence of the coding region mouse SOCS1mutXHO.

FIG. 42 is the alignment of the nucleotide and predicted amino acid sequence of the first 28 amino acids of the SH2 domain of CIS, SOCS1, SOCS2 and SOCS3, introduced nucleotide changes that lead to the R>K amino acid substitution are underlined.

FIG. 43A is a schematic representation of SOCS-1 genomic map in mice.

FIG. 43B is a schematic representation of the genomic map in mice.

FIG. 43C is a schematic representation of the SOCS-3 genomic map in mice.

FIG. 43D is a schematic representation of the SOCS-5 genomic map in mice.

FIG. 43E is a schematic representation of SOCS-9 genomic map in mice.

FIG. 43F is a schematic representation of the SOCS-11 genomic map in mice.

FIG. 44 is a diagrammatic representation of SOCS-1 targeting.

FIG. 45 is a diagrammatic representation of SOCS-2 targeting.

FIG. 46 is a diagrammatic representation of SOCS-3 targeting.

FIG. 47 is a diagrammatic representation of CIS targeting.

FIG. 48 is a diagrammatic representation of pEF-GFP-I/m SOCS-1.

FIG. 49 is a representation showing nucleotide sequences of oligonucleotides and full length coding sequence of SOCS-1.

FIG. 50 is a representation of amino acid sequence of SOCS-13.

FIG. 51 is a (A) nucleotide of mouse SOCS-5. The predicted translational start and stop sites underlined; (B) predicted amino acid sequence of mouse SOCS-5. The conventional three-letter code for amino acids is used.

FIG. 52 is a (A) nucleotide of mouse SOCS-9. The predicted translational start and stop sites underlined; (B) predicted amino acid sequence of mouse SOCS-9. The conventional three-letter code for amino acids is used.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides a new family of modulators of signal transduction. As the initial members of this family suppressed cytokine signalling, the family is referred to as the “suppressors of cytokine signalling” family of “SOCS”. The SOCS family is defined by the presence of a C-terminal domain referred to as a “SOCS box”. Different classes of SOCS molecules are defined by a motif generally but not exclusively located N-terminal to the SOCS box and which is involved by protein:molecule interaction such as protein:DNA or protein:protein interaction. Particularly preferred motifs are selected from an SH2 domain, WD-40 repeats and ankyrin repeats.

WD-40 repeats were originally recognised in the β-subunit of G-proteins. WD-40 repeats appear to form a β-propeller-like structure and may be involved in protein-protein interactions. Ankyrin repeats were originally recognised in the cytoskeletal protein ankryin.

Members of the SOCS family may be identified by any number of means. For example, SOCS1 to SOCS3 were identified by their ability to suppress cytokine-mediated signal transduction and, hence, were identified based on activity. SOCS4 to SOCS15 were identified as nucleotide sequences exhibiting similarity at the level of the SOCS box.

The SOCS box is a conserved motif located in the C-terminal region of the SOCS molecule. In accordance with the present invention, the amino acid sequence of the SOCS box is:

X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆[X_(i)]_(n)X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃[Xj]_(n)X₂₄X₂₅X₂₆X₂₇X₂₈

wherein:

X₁ is L, I, V, M, A or P;

X₂ is any amino acid residue;

X₃ is P, T or S;

X₄ is L, I, V, M, A or P;

X₅ is any amino acid;

X₆ is any amino acid;

X₇ is L, I, V, M, A, F, Y or W;

X₈ is C, T or S;

X₉ is R, K or H;

X₁₀ is any amino acid;

X₁₁ is any amino acid;

X₁₂ is L, I, V, M, A or P;

X₁₃ is any amino acid;

X₁₄ is any amino acid;

X₁₅ is any amino acid;

X₁₆ is L, I, V, M, A, P, G, C, T or S;

[X_(i)]_(n) is a sequence of n amino acids wherein n is from 1 to 50 amino acids and wherein the sequence X_(i) may comprise the same or different amino acids selected from any amino acid residue;

X₁₇ is L, I, V, M, A or P;

X₁₈ is any amino acid;

X₁₉ is any amino acid;

X₂₀ is L, I, V, M, A or P;

X₂₁ is P;

X₂₂ is L, I, V, M, A, P or G;

X₂₃ is P or N;

[X_(j)]_(n) is a sequence of n amino acids wherein n is from 0 to 50 amino acids and wherein the sequence X_(j) may comprise the same or different amino acids selected from any amino acid residue;

X₂₄ is L, I, V, M, A or P;

X₂₅ is any amino acid;

X₂₆ is any amino acid;

X₂₇ is Y or F; and

X₂₈ is L, I, V, M, A or P.

As stated above and in accordance with the present invention, SOCS proteins are divided into separate classes based on the presence of a protein:molecule interacting region such as but not limited to an SH2 domain, WD-40 repeats and ankyrin repeats located N-terminal of the SOCS box. The latter three domains are protein:protein interacting domains.

Examples of SH2 containing SOCS proteins include SOCS1, SOCS2, SOCS3, SOCS5, SOCS9, SOCS11 and SOCS14. Examples of SOCS containing WD-40 repeats include SOCS4, SOCS6 and SOCS15. Examples of SOCS containing ankyrin repeats include SOCS7, SOCS10 and SOCS12.

The present invention provides inter alia nucleic acid molecules encoding SOCS proteins, purified naturally occurring SOCS proteins as well as recombinant forms of SOCS proteins and methods of modulating signal transduction by modulating activity of SOCS proteins or expression of SOCS genes. Preferably, signal transduction is mediated by a cytokine, examples of which include EPO, TPO, G-CSF, GM-CSF, IL-3, IL-2, IL-4, IL-7, IL-13, IL-6, LIF, IL-12, IFNγ, TNFα, IL-1 and/or M-CSF. Particularly preferred cytokines include IL-6, LIF, OSM, IFN-γ and/or thrombopoietin.

Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a protein or a derivative, homologue, analogue or mimetic thereof or comprises a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. wherein said protein comprises a SOCS box in its C-terminal region and optionally a protein:molecule interacting domain N-terminal of the SOCS box.

Preferably, the protein:molecule interacting domain is a protein:DNA or protein:protein interacting domain. Most preferably, the protein:molecule interacting domain is one of an SH2 domain, WD-40 repeats and/or ankyrin repeats.

As stated above, preferably the subject SOCS modulate cytokine-mediated signal transduction. The present invention extends, however, to SOCS molecules modulating other effector-mediated signal transduction such as mediated by other endogenous or exogenous molecules, antigens, microbes and microbial products, viruses or components thereof, ions hormones and parasites. Endogenous molecules in this context are molecules produced within the cell carrying the SOCS molecule. Exogenous molecules are produced by other cells or are introduced to the body.

Preferably, the nucleic acid molecule or SOCS protein is in isolated or purified form. The terms “isolated” and “purified” means that a molecule has undergone at least one purification step away from other material.

Preferably, the nucleic acid molecule is in isolated form and is DNA such as cDNA or genomic DNA. The DNA may encode the same amino acid sequence as the naturally occurring SOCS or the SOCS may contain one or more amino acid substitutions, deletions and/or additions. The nucleotide sequence may correspond to the genomic coding sequence (including exons and introns) or to the nucleotide sequence in cDNA from mRNA transcribed from the genomic gene or it may carry one or more nucleotide substitutions, deletions and/or additions thereto.

In a preferred embodiment, the nucleic acid molecule comprises a sequence of nucleotide encoding or complementary to a sequence encoding a SOCS protein or a derivative, homologue, analogue or mimetic thereof wherein the amino acid sequence of said SOCS protein is selected from SEQ ID NO:4 (mSOCS1), SEQ ID NO:6 (mSOCS2), SEQ ID NO:8 (mSOCS3), SEQ ID NO:10 (hSOCS1), SEQ ID NO:12 (rSOCS1), SEQ ID NO:14 (mSOCS4), SEQ ID NO:18 (mSOCS5), SEQ ID NO:21 (SOCS6), SEQ ID NO:25 (mSOCS27), SEQ ID NO:29 (mSOCS8), SEQ ID NO:36 (hSOCS11), SEQ ID NO:41 (mSOCS13), SEQ ID NO:44 (mSOCS14), SEQ ID NO:46 (mSOCS15) and SEQ ID NO:48 (mSOCS15) or encodes an amino acid sequence with a single or multiple amino acid substitution, deletion and/or addition to the listed sequences or is a nucleotide sequence capable of hybridizing to the nucleic acid molecule under low stringency conditions at 42° C. In an even more preferred embodiment, the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a SOCS protein or a derivative, homologue, analogue or mimetic thereof wherein the nucleotide sequence is selected from a nucleotide sequence substantially set forth in SEQ ID NO:3 (mSOCS1), SEQ ID NO:5 (mSOCS2), SEQ ID NO:7 (mSOCS3), SEQ ID NO:9 (hSOCS11), SEQ ID NO:11 (rSOCS1), SEQ ID NO:13 (mSOCS4), SEQ ID NO:15 and SEQ ID NO:16 (hSOCS4), SEQ ID NO:17 (mSOCS5), SEQ ID NO:19 (hSOCS5), SEQ ID NO:20 (mSOCS6), SEQ ID NO:22 and SEQ ID NO:23 (hSOCS6), SEQ ID NO:24 (mSOCS7), SEQ ID NO:26 and SEQ ID NO:27 (hSOCS7), SEQ ID NO:28 (mSOCS8), SEQ ID NO:30 (mSOCS9), SEQ ID NO:31 (hSOCS9), SEQ ID NO:32 (mSOCS10), SEQ ID NO:33 and SEQ ID NO:34 (hSOCS10), SEQ ID NO:35 (hSOCS11), SEQ ID NO:37 (mSOCS12), SEQ ID NO:38 and SEQ ID NO:39 (hSOCS12), SEQ ID NO:40 (mSOCS13), SEQ ID NO:42 (hSOCS13), SEQ ID NO:43 (mSOCS14), SEQ ID NO:45 (mSOCS15) and SEQ ID NO:47 (hSOCS15) or a nucleotide sequence having at least about 15% similarity to all or a region of any of the listed sequences or a nucleic acid molecule capable of hybridizing to any of the listed sequences under low stringency conditions at 42° C.

Reference herein to a low stringency at 42° C. includes and encompasses from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1M to at least about 2M salt for hybridisation, and at least about 1M to at least 2M salt for washing conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01M to at least about 0.15M salt for hybridisation, and at least about 0.01M to at least about 0.15M salt for washing conditions.

In another embodiment, the present invention is directed to a SOCS protein or derivative, homologue, analogue or mimetic thereof wherein said SOCS protein is identified as follows:

human SOCS4 characterized by EST81149, EST180909, EST182619, ya99H09, ye70co4, yh53c09, yh77g11, yh87h05, yi45h07, yj04e06, yq12h06, yq56a06, yq60e02, yq92g03, yq97h06, yr90f01, yt69c03, y30a08, yv55f07, yv57h09, yv87h02, yv98e11, yw68d10, yw82a03, yx08a07, yx72h06, yx76b09, yy37h08, yy66b02, za81f08, zb18f07, zc06e08, zd14g06, zd51h12, zd52b09, ze25g11, ze69f02, zf54f03, zh96c07, zv66h12, zs83a08 and zs83g08;

mouse SOCS-4 characterised by mc65f04, mf42e06, mp10c10, mr81g09, and mt19h12;

human SOCS-5 characterised by EST15B103, EST15B105, EST27530 and zf50f01;

mouse SOCS-5 characterised by mc55a01, mh98f0, my26h12 and ve24e06;

human SOCS-6 characterised by yf61e08, yf93a09, yg05f12, yg41f04, yg45c02, yh11f10, yh13b05, zc35a12, ze02h08, zl09a03, zl69e10, zn39d08 and zo39e06;

mouse SOCS-6 characterised by mc04c05, md48a03, mf31d03, mh26b07, mh78e11, mh88h09, mh94h07, mi27h04 and mj29c05, mp66g04, mw75g03, va53b05, vb34h02, vc55d07, vc59e05, vc67d03, vc68d10, vc97h01, vc99c08, vd07h03, vd08c01, vd09b12, vd19b02, vd29a04 and vd46d06;

human SOCS-7 characterised by STS WI30171, EST00939, EST12913, yc29b05, yp49f10, zt10f03 and zx73g04;

mouse SOCS-7 characterised by mj39a01 and vi52h07;

mouse SOCS-8 characterised by mj6e09 and vj27a029;

human SOCS-9 characterised by CSRL-82f2-u, EST114054, yy06b07, yy06g06, zr40c09, zr72h01, yx92c08, yx93b08 and hfe0662;

mouse SOCS-9 characterised by me65d05;

human SOCS-10 characterised by aa48h10, zp35h01, zp97h12, zq08h01, zr34g05, EST73000 and HSDHEI005;

mouse SOCS-10 characterised by mb14d12, mb40f06, mg89b11, mq89e12, mp03g12and vh53c11;

human SOCS-11 characterised by zt24h06 and zr43b02;

human SOCS-13 characterised by EST59161;

mouse SOCS-13 characterised by ma39a09; me60c05, mi78g05, mk10c11, mo48g12, mp94a01, vb57c07 and vh07c11; and

human SOCS-14 characterised by mi75e03, vd29h11 and vd53g07; or a derivative or homologue of the above ESTs characterised by a nucleic acid molecule being capable of hybridizing to any the listed ESTs under low stringency conditions at 42° C.

In another embodiment, the nucleotide sequence encodes the following amino acid sequence:

X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆[X_(i)]_(n)X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃[X_(j)]_(n)X₂₄X₂₅X₂₆X₂₇X₂₈

wherein:

X₁ is L, I, V, M, A or P;

X₂ is any amino acid residue;

X₃ is P, T or S;

X₄ is L, I, V, M, A or P;

X₅ is any amino acid;

X₆ is any amino acid;

X₇ is L, I, V, M A, F, Y or W;

X₈ is C, T or S;

X₉ is R, K or H;

X₁₀ is any amino acid;

X₁₁ is any amino acid;

X₁₂ is L, I, V, M, A or P;

X₁₃ is any amino acid;

X₁₄ is any amino acid;

X₁₅ is any amino acid;

X₁₆ is L, I, V, M, A, P, G, C, T or S;

[X_(i)]_(n) is a sequence of n amino acid wherein n is from 1 to 50 amino acids and wherein the sequence X_(i) may comprise the same or different amino acids selected from any amino acid residue;

X₁₇ is L, I, V, M, A or P;

X₁₈ is any amino acid;

X₁₉ is any amino acid;

X₂₀ L, I, V, M, A or P;

X₂₁ is P;

X₂₂ is L, I, V, M, A, P or G;

X₂₃ is P or N;

[X_(j)]_(n) is a sequence of n amino acids wherein n is from 0 to 50 amino acids and wherein the sequence X_(j) may comprise the same or different amino acids selected from any amino acid residue;

X₂₄ is L, I, V, M, A or P;

X₂₅ is any amino acid;

X₂₆ is any amino acid;

X₂₇ is Y or F; and

X₂₈ is L, I, V, M, A or P.

The above sequence comparisons are preferably to the whole molecule but may also be to part thereof. Preferably, the comparisons are made to a contiguous series of at least about 21 nucleotides or at least about 5 amino acids. More preferably, the comparisons are made against at least about 21 contiguous nucleotides or at least 7 contiguous amino acids.

Comparisons may also only be made to the SOCS box region or a region encompassing the protein:molecule interacting region such as the SH2 domain WD-40 repeats and/or ankyrin repeats.

Still another embodiment of the present invention contemplates an isolated polypeptide or a derivative, homologue, analogue or mimetic thereof comprising a SOCS box in its C-terminal region.

Preferably the polypeptide further comprises a protein:molecule interacting domain such as a protein:DNA or protein:protein interacting domain. Preferably, this domain is located N-terminal of the SOCS box. It is particularly preferred for the protein:molecule interacting domain to be at least one of an SH2 domain, WD-40 repeats and/or ankyrin repeats.

Preferably, the signal transduction is mediated by a cytokine selected from EPO, TPO, G-CSF, GM-CSF, IL-3, IL-2, IL-4, IL-7, IL-13, IL-6, LIF, IL-12, IFNγ, TNFα, IL-1 and/or M-CSF. Preferred cytokines are IL-6, LIF, OSM, IFN-γ or thrombopoietin.

More preferably, the protein comprises a SOCS box having the amino acid sequence:

X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆[X_(j)]_(n) X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃[X_(j)]_(n)X₂₄X₂₅X₂₆X₂₇X₂₈

wherein

X₁ is L, I, V, M, A or P;

X₂ is any amino acid residue;

X₃ is P, T or S;

X₄ is L, I, V, M, A or P;

X₅ is any amino acid;

X₆ is any amino acid;

X₇ is L, I, V, M, A, F, Y or W;

X₈ is C, T or S;

X₉ is R, K or H;

X₁₀ is any amino acid;

X₁₁ is any amino acid;

X₁₂ is L, I, V, M, A or P;

X₁₃ is any amino acid;

X₁₄ is any amino acid;

X₁₅ is any amino acid;

X₁₆ is L, I, V, M, A, P, G, C, T or S;

[X_(i)]_(n) is a sequence of n amino acids wherein n is from 1 to 50 amino acids and wherein the sequence X_(i) may comprise the same or different amino acids selected from any amino acid residue;

X₁₇ is L, I, V, M, A or P;

X₁₈ is any amino acid;

X₁₉ is any amino acid;

X₂₀ L, I, V, M, A or P;

X_(21 is P;)

X₂₂ is L, I, V, M, A, P or G;

X₂₃ is P or N;

[X_(j)]_(n) is a sequence of n amino acids wherein n is from 0 to 50 amino acids and wherein the sequence X_(j) may comprise the same or different amino acids selected from any amino acid residue;

X₂₄ is L, I, V, M, A or P;

X₂₅ is any amino acid;

X₂₆ is any amino acid;

X₂₇ is Y or F; and

X₂₈ is L, I, V, M, A or P.

Still another embodiment provides an isolated polypeptide or a derivative, homologue, analogue or mimetic thereof comprising a sequence of amino acids substantially as set forth in SEQ ID NO:4 (mSOCS1), SEQ ID NO:6 (mSOCS2), SEQ ID NO:8 (mSOCS3), SEQ ID NO:10 (hSOCS1), SEQ ID NO:12 (rSOCS1), SEQ ID NO:14 (mSOCS4), SEQ ID NO:18 (mSOCS5), SEQ ID NO:21 (mSOCS6), SEQ ID NO:25 (mSOCS7), SEQ ID NO:29 (mSOCS8), SEQ ID NO:36 (hSOCS11), SEQ ID NO:41 (mSOCS13), SEQ ID NO:44 (mSOCS14), SEQ ID NO:46 (mSOCS15) and SEQ ID NO:48 (hSOCS15) or an amino acid sequence having at least 15% similarity to all or a part of the listed sequences.

Preferred nucleotide percentage similarities include at least about 20%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or above such as 93%, 95%, 98% or 99%.

Preferred amino acid similarities include at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97% or 98% or above.

As state above, similarity may be measured against an entire molecule or a region comprising at least 21 nucleotides or at least 7 amino acids. Preferably, similarity is measured in a conserved region such as SH2 domain, WD-40 repeats, ankyrin repeats or other protein:molecule interacting domains or a SOCS box.

The term “similarity” includes exact identity between sequences or, where the sequence differs, different amino acids are related to each other at the structural, functional, biochemical and/or conformational levels.

The nucleic acid molecule may be isolated from any animal such as humans, primates, livestock animals (e.g. horses, cows, sheep, donkeys, pigs), laboratory test animals (e.g. mice, rats, rabbits, hamsters, guinea pigs), companion animals (e.g. dogs, cats) or captive wild animals (e.g. deer, foxes, kangaroos).

The terms “derivatives” or its singular from “derivative” whether in relation to a nucleic acid molecule or a protein includes parts, mutants, fragments and analogues as well as hybrid or fusion molecules and glycosylation variants. Particularly useful derivatives comprise single or multiple amino acid substitutions, deletions and/or additions to the SOCS amino acid sequence.

Preferably, the derivatives have functional activity or alternatively act as antagonists or agonists. The present invention further extends to homologues of SOCS which include the functionally or structurally related molecule from different animal species. The present invention also encompasses analogues and mimetics. Mimetics include a class of molecule generally but not necessarily having non-amino acid structure and which functionally are capable of acting in an analogous manner to the protein for which it is a mimic, in this case, a SOCS. Mimetics may comprise a carhohydrate, aromatic ring, lipid or other complex chemical structure or may also be proteinaceous in composition. Mimetics as well as agonists and antagonists contemplated herein are conveniently located through systematic searching of environments, such as coral, marine and freshwater river beds, flora and microorganisms. This is sometimes referred to as natural product screening. Alternatively,, libraries of synthetic chemical compounds may be screened for potentially useful molecules.

The present invention further extends to a range of deletion mutants such as SOCS molecules carrying deletion in the carboxy terminal region, the amino terminal region and in both the carboxy and amino terminal regions. Molecules are also contemplated by the present invention which encompasses only the carboxy terminal region or amino terminal region or fused to another peptide, polypeptide or protein. Molecules comprising the amino terminal portion of the SOCS molecules are particularly useful as molecules capable of interacting with cytokines. For example, the N-terminal region of SOCS-1 is critical for inhibition of MI macrophage differentiation and LIF and IL-6 signalling.

As state above, the present invention contemplates agonists and antagonists of the SOCS. One example of an antagonist is an antisense oligonucleotide sequence. Useful oligonucleotides are those which have a nucleotide sequence complementary to at least s portion of the protein-coding or “sense” sequence of the nucleotide sequence. These antisense nucleotides can be used to effect the specific inhibition of gene expression. The antisense approach can cause inhibition of gene expression apparently by forming an anti-parallel duplex by complementary base pairing between the antisense construct and the targeted mRNA, presumably resulting in hybridisation arrest of translation. Ribozymes and co-suppression molecules may also be used. Antisense and other nucleic acid molecules may first need to be chemically modified to permit penetration of cell membranes and/or to increase their serum half life or otherwise make them more stable for in vivo administration. Antibodies may also act as either antagonists or agonists although are more useful in diagnostic applications or in the purification of SOCS proteins. Antagonists and agonists may also be identified following natural product screening or screening of libraries of chemical compounds or may be derivatives or analogues of the SOCS molecules.

Agonists and antagonists of SOCS proteins contemplated by the present invention include carboxy-terminal and N-terminal portions of the SOCS molecule. For example, the N-terminal portion of SOCS-1 is useful in inhibiting LIF and IL-6 signalling.

Accordingly, the present invention extends to analogues of the SOCS proteins of the present invention. Analogues may be used, for example, in the treatment or prophylaxis of cytokine mediated dysfunction such as autoimmunity, immune suppression or hyperactive immunity or other condition including but not limited to dysfunctions in the haemopoietic, endocrine, hepatic and neural systems. Dysfunctions mediated by other signal transducing elements such as hormones or endogenous or exogenous molecules, antigens, microbes and microbial products, viruses or components thereof, ions, hormones and parasites are also contemplated by the present invention.

Analogues of the proteins contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogues.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH₄; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted malemide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid, contemplated herein is shown in Table 3.

TABLE 3 Non-conventional Non-conventional amino acid Code amino acid Code α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-a-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbomyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexyalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisoleucine Nmile D-alanine Dal L-N-methylleucine Nmleu D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine Dgin L-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine Nmorn D-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine Dile L-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptopban Nmtrp D-omithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrate Mgabu D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap D-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn D-α-methylisoleucine Dmile N-amino-αmethylbutyrate Nmaabu D-α-methylleucine Dmleu a-napthylalanine Anap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln D-α-methylomithine Dmorn N-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycine Ncbut D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec D-α-methylvaline Drnval N-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhc D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg D-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine Nthr D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp D-N-methyllysine Dnmlys N-methyl-y-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet D-N-methylomithine Dnmorn N-methylcyclopentylalanine Nmcpen N-methylglycine Nala D-N-methylphenylalanine Dnrnphe N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro N-(1-methylpropyl)glycine Nile D-N-methylserine Dnrnser N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nval D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutync acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine Tbug N-(thioniethyl)glycine Ncys L-ethylglycine Etg penicillamine Pen L-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine Marg L-α-methylasparagine Masn L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug L-α-methylcysteine Mcys L-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamate Mglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet L-α-methylleucine Mleu L-α-methylysine Mlys L-α-methylmethionine Mmet L-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithine Morn L-α-methylphenylalanine Mphe L-α-methylproline Mpro L-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine 1-carboxy-1-(2,2-diphenyl- Nmbc ethylamino)cyclopropane

Crosslinkers can be used, for example, to stabilise 3D conformations, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CCH₂)_(n) spacer groups with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esteres and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of C_(α) and N_(α)-methylamino acids, introduction of double bonds between C_(α) and C_(β) atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.

These types of modifications may be important to stabilise the cytokines if administered to an individual or for use as a diagnostic reagent.

Other derivatives contemplated by the present invention include a range of glycosylation variants from a completely unglycosylated molecule to a modified glycosylated molecule. Altered glycosylation patterns may result from expression of recombinant molecules in different host cells.

Another embodiments of the present invention contemplates a method for modulating expression of a SOCS protein in a mammal, said method comprising contacting a gene encoding a SOCS or a factor/element involved in controlling expression of the SOCS gene with an effective amount of a modulator of SOCS expression for a time and under conditions sufficient to up-regulate or down-regulate or otherwise modulate expression of SOCS. An example of a modulator is a cytokine such as IL-6 or other transcription regulators of SOCS expression.

Expression includes transcription or translation or both.

Another aspect of the present invention contemplates a method of modulating activity of SOCS in a human said method comprising administering to said mammal a modulating effective amount of a molecule for a time and under conditions sufficient to increase or decrease SOCS activity. The molecule may be a proteinaceous molecule or a chemical entity and may also be a derivative of SOCS or a chemical analogue or truncation mutant of SOCS.

A further aspect of the present invention provides a method of inducing synthesis of a SOCS or transcription/translation of a SOCS comprising contacting a cell containing a SOCS gene with an effective amount of a cytokin capable of inducing said SOCS for a time and under conditions sufficient for said SOCS to be produced. For example, SOCS1 may be induced by IL-6.

Still a further aspect of the present invention contemplates a method of modulating levels of a SOCS protein in a cell said method comprising contacting a cell containing a SOCS gene with an effective amount of a modulator of SOCS gene expression or SOCS protein activity for a time and under conditions sufficient to modulate levels of said SOCS protein.

Yet a further aspect of the present invention contemplates a method of modulating signal transduction in a cell containing a SOCS gene comprising contacting said cell with an effective amount of a modulator of SOCS gene expression or SOCS protein activity for a time sufficient to modulate signal transduction.

Even yet a further aspect of the present invention contemplates a method of influencing interaction between cells wherein at least one cell carries a SOCS gene, said method comprising contacting the cell carrying the SOCS gene with an effective amount of a modulator of SOCS gene expression or SOCS protein activity for a time sufficient to modulate signal transduction.

As stated above, of the present invention contemplates a range of mimetics or small molecules capable of acting as agonists or antagonists of the SOCS. Such molecules may be obtained from natural product screening such as from coral, soil, plants or the ocean or antarctic environments. Alternatively, peptide, polypeptide or protein libraries or chemical libraries may be readily screened. For example, M1 cells expressing a SOCS do not undergo differentiation in the presence of IL-6. This system can be used to screen molecules which permit differentiation in the presence of IL-6 and a SOCS. A range of test cells may be prepared to screen for antagonists and agonists for a range of cytokines. Such molecules are preferably small molecules and may be of amino acid origin or of chemical origin. SOCS molecules interacting with signalling proteins (eg. JAKS) provide molecular screens to detect molecules which interfere or promote this interaction. Once such screening protocol involves natural product screening.

Accordingly, the present invention contemplates a pharmaceutical composition comprising SOCS or a derivative thereof or a modulator of SOCS expression or SOCS activity and one or more pharmaceutically acceptable carriers and/or diluents. These components are referred to as the “active ingredients”. These and other aspects of the present invention apply to any SOCS molecules such as but not limited to SOCS1 to SOCS15.

The pharmaceutical forms containing active ingredients suitable for injectable use include sterile aqueous solutions (where water soluble) sterile powders for the extemporaneous preparation of sterile injectable solutions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as licithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, aprabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 μg and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter. A binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compounds, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cheery or orange flavour. Of course, any material used in preparing any dosage unit from should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

The present invention also extends to forms suitable for topical application such as creams, lotions and gels.

Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such as active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.

The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 μg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 μg to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients. The effective amount may also be conveniently expressed in terms of an amount per kg of body weight. For example, from about 0.01 ng to about 10,000 mg/kg body weight may be administered.

The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carriers a nucleic acid molecule capable of modulating SOCS expression or SOCS activity. The vector may, for example, by a viral vector. In this regard, a range of gene therapies are contemplated by the present invention including isolating certain cells, genetically manipulating and returning the cell to the same subject or to a genetically related or similar subject.

Still another aspect of the present invention is directed to antibodies to SOCS and its derivatives. Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to SOCS or may be specifically raised to SOCS or derivatives thereof. In the case of the latter, SOCS or its derivatives may first need to be associated with a carrier molecule. The antibodies and/or recombinant SOCS or its derivatives of the present invention are particularly useful as therapeutic or diagnostic agents.

For example, SOCS and its derivatives can be used to screen for naturally occurring antibodies to SOCS. These may occur, for example in some autoimmune diseases. Alternatively, specific antibodies can be used to screen for SOCS. Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA. Knowledge of SOCS levels may be important for diagnosis of certain cancers or a predisposition to cancers or monitoring cytokine mediated cellular responsiveness or for monitoring certain therapeutic protocols.

Antibodies to SOCS of the present invention may be monoclonal or polyclonal. Alternatively, fragments of antibodies may be used such as Fab fragments. Furthermore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A “synthetic antibody” is considered herein to include fragments and hybrids of antibodies. The antibodies of this aspect of the present invention are particularly useful for immunotherapy and may also be used as a diagnostic tool for assessing apoptosis or monitoring the program of a therapeutic regimin.

For example, specific antibodies can be used to screen for SOCS proteins. The latter would be important, for example, as a means for screening for levels of SOCS is a cell extract or other biological fluid or purifying SOCS made by recombinant means from culture supernatant fluid. Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.

It is within the scope of this invention to include any second antibodies (monoclonal, polyclonal or fragments of antibodies or synthetic antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody. An antibody as contemplated herein includes any antibody specific to any region of SOCS.

Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein and either type is utilizable for immunoassays. The methods of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of SOCS, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques. Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.

Another aspect of the present invention contemplates a method for detecting SOCS in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for SOCS or its derivatives or homologues for a time and under conditions sufficient for an antibody SOCS complex to form and then detecting said complex.

The presence of SOCS may be accomplished in a number of ways such as by Western blotting and ELISA procedures. A wide range of immunoassay techniques are available as can be seen by reference to U.S. Pat. No. 4,016,043, 4,424,279 and 4,018,053. These, of course, include both single-site and two-site or “sandwich” assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.

Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody antigen-labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In accordance with the present invention the sample is one which might contain SOCS including cell extract, tissue biopsy or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid. The sample is, therefore, generally a biological sample comprising biological fluid but also extends to fermentation fluid and supernatant fluid such as from a cell culture.

In the typical forward sandwich assay, a first antibody having specificity for the SOCS or antigenic parts thereof, is either covalently or passively bound to a solid surface. The solid surface Is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The biding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes or overnight if more convenient) and under suitable conditions (e.g. room temperature to 37° C.) to allow binding of any subunit present in the antibody. Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hepten.

An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.

By “reporter molecule” as used in the present specification, is meant a molecule which, by its chemical nature provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminscent molecules.

In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes includes alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample. “Reporter molecule” also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.

Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope. As in the EIA, the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest. Immunofluoroescene and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.

The present invention also contemplates genetic assays such as involving PCR analysis to detect SOCS gene or its derivatives. Alternative methods or methods used in conjunction include direct nucleotide sequencing or mutation scanning such as single stranded conformation polymorphisms analysis (SSCP) as specific oligonucleotide hybridisation, as methods such as direct protein truncation tests.

Since cytokines are involved in transcription of some SOCS molecules, the detection of SOCS provides surrogate markers for cytokines or cytokine activity. This may be useful in assessing subjects with a range of conditions such as those will autoimmune diseases, for example, rheumatoid arthritis, diabetes and stiff man syndrome amongst others.

The nucleic acid molecules of the present invention may be DNA or RNA. When the nucleic acid molecule is in DNA form, it may be genomic DNA or cDNA. RNA forms of the nucleic acid molecules of the present invention are generally mRNA.

Although the nucleic acid molecules of the present invention are generally in isolated form, they may be integrated into or ligated to or otherwise fused or associated with other genetic molecules such as vector molecules and in particular expression vector molecules. Vectors and expression vectors are generally capable of replication and, if applicable, expression in one or both of a prokaryotic cell or a eukaryotic cell. Preferably, prokaryotic cells include E. coli, Bactillus sp and Pseudomonoas sp. Preferred eukaryotic cells include yeast, fungal, mammalian and insect cells.

Accordingly, another aspect of the present invention contemplates a genetic construct comprising a vector portion and a mammalian and more particularly a human SOCS gene portion, which SOCS gene portion is capable of encoding a SOCS polypeptide or a functional or immunologically interactive derivative thereof.

Preferably, the SCOS gene portion of the genetic construct is operably linked to a promoter on the vector such that said promoter is capable of directing expression of said SOCS gene portion in an appropriate cell.

In addition, the SCOS gene portion of the genetic construct may comprise all or part of the gene fused to another genetic sequence such as a nucleotide sequence encoding glutathione-S-transferase or part thereof.

The present invention extends to such genetic constructs and to prokaryotic or eukaryotic cells comprising same.

The present invention also extends to any or all derivatives of SOCS including mutants, part, fragments, portions, homologues and analogues or their encoding genetic sequence including single or multiple nucleotide or amino acid substitutions, additions and/or deletions to the naturally occurring nucleotide or amino acid sequence. The present invention also extends to mimetics and agonists and antagonists of SOCS.

The SCOS and its genetic sequence of the present invention will be useful in the generation of a range of therapeutic and diagnostic reagents and will be especially useful in the detection of a cytokine involved in a particular cellular response or a receptor for that cytokine. For example, cells expressing SOCS gene such as M1 cells expressing the SOCS1 gene, will no longer be responsive to a particular cytokine such as, in the case of SOCS1, IL-6. Clearly, the present invention further contemplates cells such as M1 cells expressing any SOCS gene such as from SOCS1 to SOCS15. Furthermore, the present invention provides the use of molecules that regulate on potentiate the ability of therapeutic cytokines. For example, molecules which block some SOCS activity, may act to potential therapeutic cytokine activity (eg. G-CSF). Soluble SOCS polypeptides are also contemplated to be particularly useful in the treatment of disease, injury or abnormally involving cytokine mediated cellular responsiveness such as hyperimmunity, immunosuppression, allergies, hypertension and the like.

A further aspect of the present invention contemplates the use of SOCS or its functional derivatives in the manufacture of a medicament for the treatment of conditions involving cytokine mediated cellular responsiveness.

The present invention further contemplates transgenic mammalian cells expressing a SOCS gene. Such cells are useful indicator cell lines for assaying for suppression of cytokine function. One example is M1 cells expressing a SOCS gene. Such cell lines may be useful for screening for cytokines or screening molecules such as naturally occurring molecules from plants, coral, microorganisms or bio-organically active soil or water capable of acting as cytokine antagonists or agonists. The present invention further contemplates transgenic animals such as mice, rats, sheep, pigs, rabbits and guinea pigs which are homozygous or heterozygous knockout animals for the SOCS genes or parts thereof.

The present invention further contemplates hybrids between different SOCS from the same or different animal species. For example, a hybrid may be formed between all or a functional part of mouse SOCS1 and human SOCS1. Alternatively, the hybrid may be between all or part of mouse SOCS1 and mouse SOS2. All such hybrids are contemplated herein and are particularly useful in developing pleiotropic molecules.

The present invention further contemplates a range of genetic based diagnostic assays screening for individuals with defective SOCS genes. Such mutations may result in cell types not being responsive to a particular cytokine or resulting in over responsiveness leading to a range of conditions. The SOCS genetic sequence can be readily verified using a range of PCR or other techniques to determine whether a mutation is resident in the gene. Appropriate gene therapy or other interventionist therapy may then be adopted.

The present invention is further described by the following non-limiting Examples.

Examples 1-16 relates to SOCS1, SOCS2 and SCOS3 which were identified on the basis of activity. Examples 17-24 relate to various aspects of SOCS4 to SOCS15 which were cloned initially on the basis of sequence similarity. Examples 25-36 relate to specific aspects of SOCS4 to SOCS15, respectively.

EXAMPLE 1 CELL CULTURE AND CYTOKINES

The M1 cell line was derived from a spontaneously arising leukaemia in SL mice [Ichikawa, 1969]. Parental MI cells used in this study have been in passage at the Walter and Eliza Hall Institute for Medical Research, Melbourne, Victoria, Australia, for approximately 10 years. M1 cells were maintained by weekly passage in Dulbecco's modified Eagle's medium (DME) containing 10% (v/v) foetal bovine serum (FCS). Recombinant cytokines are generally available from commercial sources or were prepared by published methods. Recombinant murine LIF was produced in Escherichia coli and purified, as previously described [Gearing, 1989]. Purified human oncostatin M was purchased from PeproTech Inc. (Rocky Hill, N.J. USA), and purified mouse IFN-γ was obtained from Genzyme Diagnostics (Cambridge, Mass. USA). Recombinant murine thrombopoietin was produced as a FLAGTM-tagged fusion protein in CHO cells and then purified.

EXAMPLE 2 AGAR COLONY ASSAYS

In order to assay the differentiation of M1 cells in response to cytokines, 300 cells were cultured in 35 mm Petri dishes containing 1 ml of DME supplemented with 20% (v/v) fital calf serum (FCS). 0.3% (w/v) agar and 0.1 ml of serial dilutions of IL-6, LIF, OSM, IFN-γ, tpo or dexamethasone (Sigma Chemical Company, St. Louis, Mich.). After 7 days culture at 37° C. in a fully humidified atmosphere, containing 10% (v/v) CO₂ in air, colonies of MI cells were counted and classified as differentiated if they were composed of dispersed cells or had a corona of dispersed cells around a tightly packed centre.

EXAMPLE 3 GENERATION OF RETROVIRAL LIBRARY

A cDNA expression library was constructed from the factor-dependent haemopoietic cell line FDC-P1, essentially as described [Rayner, 1994]. Briefly, cDNA was cloned into the retroviral vector pRUFneo and then transfected into an amphotrophic packaging cell line (PA317). Transiently generated virus was harvested from the cell supernatant at 48 hr posttransfection, and used to infect Y2 ecotropic packaging cells, to generate a high titre virus-producing cell line.

EXAMPLE 4 RETROVIRAL INFECTION OF M1 CELLS

Pools of 10⁶ infected Ψ2 cells were irradiated (3000 rad)and concultivated with 10⁶ M1 cells in DME supplemented with 10% (v/v) FCS and 4 μg/ml Polybrene, for 2 days at 37° C. To select for IL-6-unresponsive clones, retrovirally-infected M1 cells were washed once in DME, and cultured at approximately 2×10⁴ cells/ml in 1 ml agar cultures containing 400 μg/ml geneticin GibcoBRL, Grand Island, N.Y.) and 100 ng/ml IL-6. The efficiency of infection of MI cells was 1-2%, as estimated by agar plating the infected cells in the presence of geneticin only.

EXAMPLE 5 PCR

Genomic DNA from retrovirally-infected M1 cells was digested with Sac I and 1 μg of phenol/chloroform extracted DNA was then amplified by polymerase chain reaction (PCR). Primers used for amplification of cDNA inserts from the integrated retrovirus were GAG3 (4′CACGCCGCCCACGTGAAGGC 3′ [SEQ ID NO:1]), which corresponds to the vector gag sequence approximately 30 bp 5′ of the multiple cloning site, and HSVTK (4′ TTCGCCAATGACAAGACGCT 3′ [SEQ ID NO:2]), which corresponds to the pMCl neo sequence approximately 200 bp 3′ of the multiple cloning site. The PCR entailed an initial denaturation at 94° C. for 5 min, 35 cycles of denaturation at 94° C. for 1 min, annealing at 56° C. for 2 min. and extension at 72° C. for 3 min, followed by a final 10 min extension. PCR products were gel purified and then ligated into the pGEM-T plasmid (Promega, Madison, Wis.),and sequenced using an ABI PRISM Dye Terminator Cycle Sequencing Kit and a Model 373 Automated DNA Sequencer (Applied Biosystems Inc., Foster City, Calif.).

EXAMPLE 6 CLONING OF cDNAs

Independent cDNA clones encoding mouse SOCS1 were isolated from a murine thymus cDNA library essentially as described (Hilton et al., 1994). The nucleotide and predicted amino acid sequences of mouse SOCS1 cDNA were compared to databases using the BLASTIN and TFASTA algorithms (Pearson and Lipman, 1988; Pearson, 1990; Altsheul et al., 1990). Oligonucleotides were designed from the ESTs encoding human SOCS1 and mouse SCO-1 and SOCS3 and used to probe commercially available mouse thymus and spleen cDNA libraries. Sequencing was performed using an ABI automated sequencer according to the manufacturer's instructions.

EXAMPLE 7 SOUTHERN AND NORTHERN BLOT ANALYSES AND RT-PCR

³²P-labelled probes were generated using a random decanucleotide labelling kit (Bresated, Adelaide, South Australia) from a 600 bp Pst I fragment encoding neomycin phosphotransfease from the plasmid pPGKneo, 1070 bp fragment of the SOCS1 gene obtained by digestion of the 1.4 kbp PCR product with Xho I, SOCS2, SOCS3, CIS and a 1.2 kbp fragment of the chicken glyceraldehyde 3-phosphate dehydrogenase gene [Dugaiczyk, 1983].

Genomic DNA was isolated from cells using a proteinase K-sodium dodecyl sulfate procedure essentially as described. Fifteen micrograms of DNA was digested with either BamHI or Sac I, fractionated on a 0.8% (w/v) agarose gel, transferred to GeneScreenPlus membrane (Du Pont NEN, Boston, Mass.), prehybridised, hybridised with random-primed ³²P-labelled DNA fragments and washed essentially as described [Sambrook, 1989].

Total RNA was isolated from cells and tissues using Trizol Reagent, as recommended by the manufacturer (GibcoBRL, Gran Island, N.Y.). When required polyA+ mRNA was purified essentially as described [Alexander, 1995]. Northern blots were prehybridised, hybridized with random-primed 32P-labelled DNA fragments and washed as described [Alexander, 1995].

To assess the induction of SOCS genes by IL-6, mice (C57BL6) were injected intravenously with 5 μg IL-6 followed by harvest of the liver at the indicated timepoints after injection. MI cells were cultured in the presence of 20 ng/ml IL-6 and harvested at the indicated times. For RT-PCR analysis, bone marrow cells were harvested as described (Metacalf et al. 1995) and stimulated for 1 hr at 37° C. with 100 ng/ml of a range of cytokines. RT-PCR was performed on total RNA as described (Metcalf et al., 1995), PCR products were resolved on an agarose gel and Southern blots were hybridised with probes specific for each SOCS family member. Expression of β-actin was assesed to ensure uniformity of amplification.

EXAMPLE 8 DNA CONSTRUCTS AND TRANSFECTION

A cDNA encoding epitope-tagged SOCS1 was generated by subcloning the entire SCOS1 coding region into the pEF-BOS expression vector [Mizushima, 1990], engineered to encode an inframe FLAG epitope downstream of an initiation methionine (pF-SOCS1). Using electroporation as described previously [Hilton, 1994], M1 cells expressing the thrombopoietin receptor (M1.mpl) were transfected with the 20 μg of Aat II-digested pf-SOCS1 expression plasmid and 2 μg of a Sca I-digested plasmid in which transcription of a cDNA encoding puromycin N-acetyl transferase was driven from the mouse phosphoglycerokinase promoter (pPGKPuropA). After 48 hours in culture, transfected cells were selected with 20 μg/ml puromycin (Sigma Chemical Company, St. Louis, Mo.), and screened for expression of SCOS1 by Western blotting, using the M2 anti-FLAG monoclonal antibody according to the manufacturer's instructions (Eastman Kodak, Rochester N.Y.). In other experiments M1 cells were transfected with only the pF-SOCS1 plasmid or a control and selected by their ability to grow in agar in the presence of 100 ng/ml of IL-6.

EXAMPLE 9 IMMUNOPRECIPITATION AND WESTERN BLOTTING

Prior to either immunoprecipitaion or Western blotting, 10⁷ M1 cells or their derivatives were washed twice, resuspended in 1 ml of DME, and incubated at 37° C. for 30 min. The cells were then stimulated for 4 min at 37° C. with either saline or 100 ng/ml IL-6, after which sodium vanadate (Sigma Chemical Co., St. Louis, Mich.) was added to a concentration of 1 mM. Cells were placed on ice, washed once with saline containing 1 mM sodium vanadate, and the solubilised for 5 min on ice with 300 μl 1% (v/v) Triton X-100, 150 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl pH 7.4, containing Complete protease inhibitors (Boehringer Mannheim, Mannheim, Germany) and 1 mM sodium vanadate. Lysates were cleared by centrifugation and quantitated using a Coomassie Protein Assay Reagent (Pierce, Rockford Ill.).

For immunopecipitations, equal concentrations of protein extracts (1-2 mg) were incubated for 1 hr or overnight at 4° C. with either 4 μg of anti-gp130 antibody (M20; Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) or 8 νg of anti-phosphotyrosine antibody (4G10; Upstate Biotechnology Inc., Lake Placid N.Y.), and 15 μl packed volume of Protein G Sepharose (Pharmacia, Uppsala, Sweden) [Hilton et al., 1995]. Immunoprecipitates were washed twice in 1% (v/v) NP40, 150 mM NaCl, 50 mM tris-HCl pH 8.0, containing. Complete protease inhibitors (Boehringer Mannheim, Mannheim, German and 1 mM sodium vanadate. The samples were heated for 5 min at 95° C. in SDS sample buffer (625 mM Tris-HCl pH 6.8, 0.05% (w/v) SDS, 0.1% (v/v) glycerol, bromophenol blue, 0.125% (v/v) 2-mercaptoethanol), fractionated by SDS-PAGE and immunoblotted as described above.

For Western blotting, 10 μg of protein from a cellular extract or material from an immunoprecipitation reaction was loaded onto 4-15% Ready gels (Bio-Rad Laboratories, Hercules Calif.), and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to PVDF membrane (Micron Separations Inc. Westborough Mass.) for 1 hr at 100 V. The membranes were probed with the following primary antibodies; anti-tyrosine phosphorylated STAT3 (1:1000 dilution; New England Biolabs, Beverly, Mass.); anti-STAT3 (C-20; 1:100 dilution; Santa Cruz Biotechnology Inc., Santa Cruz Calif.); anti-gp130 (M20, 1:100 dilution; Santa Cruz Biotechnology Inc., Santa Cruz Calif.); anti-phosphotyrosine (horseradish peroxidase-conjugated RC20, 1:5000 dilution; Transduction Laboratories, Lexington, Ky.); anti-tyrosine phosphorylated MAP kinase and anti-MAP kinase antibodies (1:1000 dilution; New England Biolabs, Beverly, Mass.). Blots were visualised using peroxidase-conjugated secondary antibodies and Enhanced Chemiluminesence (ECL) reagents according to the manufacturer's instructions (Pierce, Rockford Ill.).

EXAMPLE 10 ELECTROPHORETIC MOBILITY SHIFT ASSAYS

Assays were performed as described [Novak, 1995], using the high affinity ISF (c-sis-inducible factor: binding site m67 [Wakao, 1994]. Protein extracts were prepared from M1 cells incubated for 4-10 min at 37° C. in 10 ml serum-free DME containing either saline, 100 ng/ml IL-6 or 100 ng/ml IFN-γ. The binding reactions contained 4-6 μg protein (constant within a given experiment), 5 ng ³²P-labelled m67 oligonucleotide, and 800 ng sonicated salmon sperm DNA. For certain experiments, protein samples were preincubated with an excess of unlabelled m67 olignucleotide, or antibodies specific for either STAT1 (Transduction Laboratories, Lexington, Ky.) or STAT3 (Santa Cruz Biotechnology Inc., Santa Crus Calif.) as described [Novak, 1995].

Western blots were performed using anti-tyrosine phosphorylated STAT3 or anti-STAT3 (New England Biolabs, Beverly, Mass.) or anti-gp130 (Santa Crus Biotechnology Inc.) as described (Nicola et al., 1996), EMSA were performed using the m67 oligonucleotide probe, as described (Novak et al., 1995).

EXAMPLE 11 EXPRESSIONS CLONING OF A NOVEL SUPPRESSOR OF CYTOKINE SIGNAL TRANSDUCTION

In order to identify cDNAs capable of suppressing cytokine signal transduction, an expression cloning approach was adopted. This strategy central on M1 cells, a monocytic leukaemia cell line that differentiates into mature macrophages and ceases proliferation in response to the cytokines IL-6, LIF, OSM and IFN-γ, and the steroid dexamethasone.

Parental M1 cells were infected with the RUFneo retrovirus, into which cDNAs from the factor-dependent haemopoietic cell line FDC-P1 had been cloned. In this retrovirus, transcription of both the neomycin resistance gene and the cloned cDNA was driven off the powerful constitutive promoter present in the retroviral LTR (FIG. 1).When cultured in semi-solid agar, parental M1 cells form large tightly packed colonies. Upon stimulation with IL-6, M1 cells undergo rapid differentiation, resulting in the formation in agar of only single macrophages or small dispersed clusters of cells. Retrovirally-infected M1 cells that were unresponsive to IL-6 were selected in semi-solid agar culture by their ability to form large, tightly packed colonies in the presence of IL-6 and geneticin. A single stable IL-6-unresponsive clone, 4A2, was obtained after examining 10⁴ infected cells.

A fragment of the neomycin phosphotransferase (neo) gene was used to probe a Southern blot of genomic DNA from clone 4A2 and this revealed that the cell line was infected with a single retrovirus containing a cDNA approximately 1.4 kbp in length (FIG. 2). PCR amplification using primers from the retroviral vector which flanked the cDNA cloning site enabled recovery of a 1.4 kbp cDNA insert, which we have named suppressor of cytokine signalling-1, or SOCS1. This PCR product was used to probe a similar Southern blot of 4A2 genomic DNA and hybridised to two fragments, one which corresponded to the endogenous SOCS1 gene and the other, which matched the size of the band seen using the neo probe, corresponded to the SOCS1 cDNA cloned into the integrated retrovirus. The latter was not observed in an M1 cell clone infected with a retrovirus containing an irrelevant cDNA. Similarly, Northern blot analysis revealed that SOCS1 mRNA was abundant in the cell line 4A2, but not in the control infected M1 cell clone.

EXAMPLE 12 SOCS1, SOCS2, SOCS3 AND CIS DEFINE A NEW FAMILY OF SH2-CONTAINING PROTEINS

The SOCS1 PCR product was used as a probe to isolate homologous cDNAs from a mouse thymus cDNA library. The sequence of the cDNAs proved to be identical to the PCR product, suggesting that constitutive or over expression, rather than mutation, of the SOCS1 protein was sufficient for generating an IL-6-unresponsive phenotype. Comparison of the sequence of SOCS1 cDNA with nucleotide sequence databases revealed that it was present on mouse and rat genomic DNA clones containing the protamine gene cluster found on mouse chromosome 16. Closer inspection revealed that the 1.4 kb SOCS1 sequence was not homologous to any of the protamine genes, but rather represented a previously unidentified open reading frame located at the extreme 3′ end of these clones (FIG. 2). There were no regions of discontinuity between the sequences of the SOCS1 cDNA and genomic locus, suggesting that SOCS1 is encoded by a single exon. In addition to the genomic clone containing the protamine genes, a series of murine and human expressed sequenced tags (ESTs) also revealed large blocks of nucleotide sequence identity to mouse SOCS1. The sequence information provided by the human ESTs allowed the rapid cloning of cDNAs encoding human SOCS1.

The mouse and rat SOCS1 gene encodes a 212 amino acid protein whereas the human SOCS1 gene encodes a 211 amino acid protein. Mouse, rat and human SOCS1 proteins share 95-99% amino acid identity (FIG. 3). A search of translated nucleic acid databases with the predicted amino acid sequence of SOCS1 showed that it was most related to a recently cloned cytokine-inducible immediate early gene product, CIS, and two classes of ESTs. Full length cDNAs from the two classes of ESTs were isolated and found to encode proteins of similar length and overall structure to SOCS1 and CIS. These clones were given the names SOCS2 and SOCS3. Each of the four proteins contains a central SH2 domain and a C-terminal region termed the SOCS motif. The SOCS1 proteins exhibit an extremely high level of amino acid sequence similarity (95-99% identity) amongst different species. However, the forms of the SOCS1, SOCS2, SOCS3 and CIS from the same animal, while clearly defining a new family of SH2-containing proteins, exhibited a lower amino acid identify. SOCS2 and CIS exhibit approximately 38% amino acid identity, while the remaining members of the family share approximately 25% amino acid identity (FIG. 3). The coding region of the genes for SOCS1 and SOC3 appear to contain no introns while the coding region of the genes for SOCS2 and CIS contain one and two introns, respectively.

The Genbank Accession Numbers for the sequences referred to herein are mouse SOCS1 cDNA (U88325), human SOCS1 cDNA (U88326), mouse SOCS2 cDNA (U88327), mouse SOCS3 cDNA (U88328).

EXAMPLE 13 CONSTITUTIVE EXPRESSION OF SOCS1 SUPPRESSES THE ACTION OF A RANGE OF CYTOKINES

To a formally establish that the phenotype of the 4A2 cell line was directly related to expression of SOCS1, and not to unrelated genetic changes which may have occurred independently in these cells, a cDNA encoding an epitope-tagged version of SOCS1 under the control of the EF1α promoter was transfected into parental M1 cells, and M1 cells expressing the receptor for thrombopoietin, c-mpl (M1.mpl). Transfection of the SOCS1 expression vector into both cell lines resulted in an increase in the frequency of IL-6 unresponsive M1 cells.

Multiple independent clones of M1 cells expression SOCS1, as detected by Western blot, displayed a cytokine-unresponsive phenotype that was indistinguishable from 4A2. Further, if transfectants were not maintained in puromycin, expression of SOCS1 was lost over time and cells regained their cytokine responsiveness. In the absence of cytokine, colonies derived from 4A2 and other SOCS1 expressing clones characteristically grew to a smaller size than colones formed by control M1 cells.

The effect of constitutive SOCS1 expression on the response of M1 cells to a range of cytokines was investigated using the 4A2 cell line and a clone of M1.mpl cells expressing SOCS1 (M1.mpl.SOCS1). Unlike parental M1 cells and M1.mpl cells, the two cell lines expressing SOCS1 continued to proliferate and failed to form differentiated colonies in response to either IL-6, LIF, OSM, IFN-γ or, in the case of the M1.mpl.SOCS1 cell line, thrombopoietin. For both cell lines, however, a normal response to dexamethasone was observed, suggesting that SOCS1 specifically affected cytokine signal transduction rather than differentiation per se. Consistent with these data, while parental M1 cells and M1.mpl cells became large and vacuolated in response to IL-6, 4A2 and M1.mpl.SOCS1 cells showed no evidence of morphological differentiation in response to IL-6 or other cytokines.

EXAMPLE 14 SOCS1 INHIBITS A RANGE OF IL-6 SIGNAL TRANSDUCTION PROCESSES, INCLUDING STAT3 PHOSPHORYLATION AND ACTIVATION

Phosphorylation of the cell surface receptor component gp130, the cytoplasmic tyrosine kinase JAK1 and the transcription factor STAT3 is thought to play a central role in IL-6 signal transduction. These events were compared in the parental M1 and M1.mpl cell lines and their SOCS1-expressing counterparts. As expected, gp130 was phosphorylated rapidly in response to IL-6 in both parental lines, however, this was reduced five- to ten-fold in the cell lines expressing SOCS1. Likewise, STAT3 phosphorylation was also reduced by approximately ten-fold in response to IL-6 in those cell lines expressing SOCS1. Consistent with a reduction in STAT3 phosphorylation, activation of specific STAT DNA binding complexes, as determined by electrophoretic mobility shift assay, was also reduced. Notably, there was a reduction in the formation of SIF-A (containing STAT3), SIF-B (STAT1/STAT3 heterodimer) and SIF-C (containing STAT1), the three STAT complexes induced in M1 cells stimulated with IL-6. Similarly, constitutive expression of SOCS1 also inhibited IFN-γ-stimulated formation of p91 homodimers. STAT phosphorylation and activation were not the only cytoplasmic processes to be effected by SOCS1 expression, as the phosphorylation of other proteins, including shc and MAP kinase, was reduced to a similar extent.

EXAMPLE 15 TRANSCRIPTION OF THE SOCS1 GENE IS STIMULATED BY IL-6 IN VITRO AND IN VIVO

Although SOCS1 can inhibit cytokine signal transduction when constitutively expressed in M1 cells, this does not necessarily indicate that SOCS1 normally functions to negatively regulate an IL-6 response. In order to investigate this possibility the inventors determined whether transcription of the SOCS1 gene is regulated in the response of M1 cells to IL-6 and, because of the critical role IL-6 plays in regulating the acute phase response to injury and infection, the response of the liver to intravenous injection of 5 mg IL-6. In the absence of IL-6, SOCS1 mRNA was undetectable in either M1 cells or in the liver. However, for both cell types, a 1.4 kb SOCS1 transcript was induced within 20 to 40 minutes by IL-6. For M1 cells, where the IL-6 was present throughout the experiment, the level of SOCS1 mRNA remained elevated. In contrast, IL-6 was administered in vivo by a single intravenous injection and was rapidly cleared from the circulation, resulting in a pulse of IL-6 stimulation to the lever. Consistent with this, transient expression of SOCS1 mRNA was detectable in the lever, peaking approximately 40 minutes after injection and declining to basal levels within 4 hours.

EXAMPLE 16 REGULATION OF SOCS GENES

Since CIS was cloned as a cytokine-inducible immediate early gene the inventors examined whether SOCS1, SOCS2 and SOCS3 were similarly regulated. The basal pattern of expression of the four SOCS genes was examined by Northern blot analysis of mRNA from a variety of tissues from male and female C57B1/6 mice. Constitutive expression of SOCS1 was observed in the thymus and to a lesser extend in the spleen and the lung. SOCS2 expression was restricted primarily to the testis and in some animals the liver and lung; for SOCS3 a low level of expression was observed in the lung, spleen and thymus, while CIS expression was more widespread, including the testis, heart, lung, kidney and, in some animals, the liver.

The inventors sought to determine whether expression of the four SOCS genes was regulated by IL-6. Northern blots of mRNA prepared from the livers of untreated and IL-6-injected mice, or from unstimulated and IL-6-stimulated M1 cells, were hybridised with labelled fragments of SOCS1, SOCS2, SOCS3 and CIS cDNAs. Expression of all four SOCS genes was increased in the liver following IL-6 injection, however the kinetics of induction appeared to differ. Expression of SOCS1 and SOCS3 was transient in the liver, with mRNA detectable after 20 minutes of IL-6 injection and declining to basal levels within 4 hours for SOCS and 8 hours for SOCS3. Induction of SOCS2 and CIS mRNA in the lever followed similar initial kinetics to that of SOCS1, but was maintained at an elevated level for at least 24 hours. A similar induction of SOCS gene mRNA was observed in other organs, notably the lung and the spleen. In contrast, in M1 cells, while SOCS1 and CIS mRNA were induced by IL-6, no induction of either SOCS2 or SOCS3 expression was detected. This result highlights cell type-specific differences in the expression of the genes of SOCS family members in response to the same cytokine.

In order to examine the spectrum of cytokines that was capable of inducing transcription of the various members of the SOCS gene family, bone marrow cells were stimulated for an hour with a range of cytokines, after which mRNA was extracted and cDNA was synthesised. PCR was then used to assess the expression of SOCS1, SOCS2, SOCS3 and CIS. In the absence of stimulation, little or no expression of any of the SOCS genes was detectable in bone marrow by PCR. Stimulation of bone marrow cells with a broad array of cytokines appeared capable of up regulating mRNA for one or more members of the SOCS family. IFNγ, for example, induced expression of all four SOCS genes, while erythropoietin, granulocyte colony-stimulating factor, granulocyte-macrophage colony stimulating factor and interleukin-3 induced expression of SOCS2, SOCS3 and CIS. Interestingly, tumor necrosis factor alpha, macrophage colony-stimulating factor and interleukin-1, which act through receptors that do not fall into the type I cytokine receptor class also appeared capable of inducing expression of SOCS3 and CIS, suggesting that SOCS proteins may play a broader role in regulating signal transduction.

As constitutive expression of SOCS1 inhibited the response of M1 cells to a range of cytokines, the inventors examined whether phosphorylation of the cell surface receptor component gp130 and the transcription factor STAT3, which are though to play a central role in IL-6 signal transduction, were affected. These events were compared in the parental M1 and M1.mpl cell lines and their SOCS1-expressing counterparts. As expected, gp130 was phyosphorylated rapidly in response to IL-6 in both parental lines, however, this was reduced in the cell lines expressing SOCS1. Likewise, STAT3 phosphorylation was also reduced in response to IL-6 in those cell lines expressing SOCS1. Consistent with a reduction in STAT3 phosphorylation, activation of specific STAT/DNA binding complexes, as determined by electrophoretic mobility shift assay, was also reduced. Notably, there was a failure to form SIF-A (containing STAT3) and SIF-B(STAT1/STAT3 heterodimer), the major STAT complexes induced in M1 cells stimulated with IL-6. Similarly, constitutive expression of SOCS1 also inhibited IFNγ-stimulating formation of SIF-C(STAT1 homodimer; FIG. 12B). These experiments are consistent with the proposal that SOCS1 inhibits signal transduction upstream of receptor and STAT phosphorylation, potentially at the level of the JAK kinases.

The ability of SOCS1 to inhibit signal transduction and ultimately the biological response to cytokines suggest that, like the SH2-containing phosphatase SHF-1 [Ihle et al, 1994; Yi et al, 1993], the SOCS proteins may play a central role in controlling the intensity and/or duration of a cell's response to a diverse range of extracellular stimuli by suppressing the signal transduction process. The evidence provided here indicates that the SOCS family acts in a classical negative feedback loop for cytokine signal transduction. Like other genes such as OSM, expression of genes encoding the SOCS proteins is induced by cytokines through the activation of STATs. Once expressed, it is proposed that the SOCS proteins inhibit the activity of JAKs and so reduce the phosphorylation of receptors and STATs, thereby suppressing signal transduction and any ensuing biological response. Importantly, inhibition of STAT activation will, over time, lead to a reduction in SOCS gene expression, allowing cells to regain responsiveness to cytokines.

EXAMPLE 17 DATABASE SEARCHES

The NCBI genetic sequence database (Genbank), which encompasses the major database of expressed sequence tags (ESTs) and TIGR database of human expressed sequence tags, were searched for sequences with similarity to a concensus SOCS box sequence using the TFASTA and MOTIF/PATTERN algorithms [Pearson, 1990; Cockwell and Giles, 1989]. Using the software package SRS [Etzold et al, 1996], ESTs that exhibited similarity to the SOCS box (and their partners derived from sequencing the other end of cDNAs) were retrieved and assembled into contigs using Autoassembler (Applied Biosystems, Foster City, Calif.). Consensus nucleotide sequences derived from overlapping ESTs were then used to search the various databases using BLASTN [Altschul et al, 1990]. Again, positive ESTs were retrieved and added to the contig. This process was repeated until no additional ESTs could be recovered. Final consensus nucleotide sequences were then translated using Sequence Navigator (Applied Biosystems, Foster City, Calif.).

The ESTs encoding the new SOCS proteins are as follows: human SOCS4 (EST81149, EST180909, EST182619, ya99H09, ye70co4, yh53c09, yh77g11, yh87h05, yi45h07, yj04e06, yq12h06, yq56a06, yq60e02, yq92g03, yq97h06, yr90f01, yt69c03, yv30a08, yv55f07, yv57h09, yv87h02, yv98e11, yw68d10, yw82a03, yx08a07, yx72h06, yx76b09, yy37h08, yy66b02, za81f08, zb18f07, zc06e08, zd14g06, zd51h12, zd52b09, ze25g11, ze69f02, zf54f03, zh96e07, zv66h12, zs83a08 and zs83g08). mouse SOCS-4 (mc65f04, mf42e06, mp10c10, mr81g09, and mt19h12). human SOCS-5 (EST15B103, EST15B105, EST27530 and zf50f01). mouse SOCS-5 (mc55a01, mh98f09, my26h12 and ve24e06). human SOCS-6 (yf61e08, yf93a09, yg05f12, yg41f04, yg45c02, yh11f10, yh13b05, zc35a12, ze02h08, zl09a03, zl69e10, zn39d08 and zo39e06). mouse SOCS-6 (mc04c05, md48a03, mf31d03, mh26b07, mh78e11, mh88h09, mh94h07, mi27h04 and mj29c05, mp66g04, mw75g03, va53b05, vb34h02, vc55d07, vc59e05, vc67d03, vc68d10, vc97h01, vc99c08, vd07h03, vd08c02, vd09b12, vd19b02, vd29a04 and vd46d06). human SOCS-7 (STS WI30171, EST00939, EST12913, yc29b05, yp49f10, zt10f03 and zx73g04). mouse SOCS-7 (mj39a01 and vi52h07). mouse SOCS-8 (mj6e09 and vj27a029). human SOCS-9 (CSRL-82f2-u, EST114054, yy06b07, yy06g06, zr40c09, zr72h01, yx92c08, yx93b08 and hef0662). mouse SOCS-9 (me65d05). human SOCS-10(ss48h10, zp35h01, zp97h12, zq08h01, zr34g05, EST73000 and HSDHEI005). mouse SOCS-10 (mb14d12, mb40f06, mg89b11, mq89e12, mp03g12 and vh53c11). human SOCS-11 (zt24h06 and zr43b02). human SOCS-13 (EST5916). mouse SOCS-13 (ma39a09, me60c05, mi78g05, mk10c11, mo48g12, mp94a01, vb57c07 and vh07c11). human SOCS-14 (mi75e03, vd29h11 and vd53g07).

EXAMPLE 18 cDNA CLONING

Based on the consensus sequences derived from overlapping ESTs, oligonucleotides were designed that were specific to various members of the SOCS family. As described above, oligonucleotides were labelled and used to screen commercially available genomic and cDNA libraries cloned with λ bacteriophage. Genomic and/or cDNA clones covering the entire coding region of mouse SOCS4, mouse SOCS5 and mouse SOCS6 were isolated. The entire gene for SOCS15 is on the human 12p13 BAC (Genbank Accession Number HSU47924) and the mouse chromosome 6 BAC (Genbank Accession Number AC002393). Partial cDNAs for mouse SOCS7, SOCS9, SOCS10, SOCS11, SOCS12, SOCS13 and SOCS14 were also isolated.

EXAMPLE 19 NORTHERN BLOTS AND rtPCR

Northern blots were performed as described above. The source of hybridisation probes were as follows; (i) the entire coding region of the mouse SOCS1 cDNA, (ii) a 1059 bp PCR product derived from coding region of SOCS5 upstream of the SH2 domain, (iii) the entire coding region of the mouse SOCS6 cDNA, (iv) a 790 bp PCR product derived from the coding region of a partial SOCS7 cDNA and (v) and 1200 bp PsI I fragment of the chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA.

EXAMPLE 20 ADDITIONAL MEMBERS OF SOCS FAMILY

SOC1, SOCS2 and SOCS3 are members of the SOCS protein family identified in Examples 1-16. Each contains a central SH2 domain and a conserved motif at the C-terminus, named the SOCS box. In order to isolate further members of this protein family, various DNA databases were searched with the amino acid sequence corresponding to conserved residues of the SOCS box. This search revealed the presence of human and mouse ESTs encoding twelve further members of the SOCS protein family (FIG. 4). Using this sequence information cDNAs encoding SOCS4, SOCS5, SOCS6, SOCS7, SOCS9, SOCS10, SOCS11, SOCS12, SOCS13, SOCS14 and SOCS15 have been isolated. Further analysis of contigs derived from ESTs and cDNAs revealed that the SOCS proteins could be placed into three groups according to their predicted structure N-terminal of the SOCS box. The three groups are those with (i) SH2 domains, (ii) WD-40 repeats and (iii) ankyrin repeats.

EXAMPLE 21 SOCS PROTEIN WITH SH2 DOMAINS

Eight SOCS proteins with SH2 domains have been identified. These include SOCS1, SOCS2 and SOCS3, SOCS5, SOCS9, SOCS11 and SOCS14 (FIG. 4). Full length cDNAs were isolated for mouse SOCS5 and SOCS14 and partial clones encoding mouse SOCS9 and SOCS14. Analysis of primary amino acid sequence and genomic structure suggest that pairs of these proteins (SOCS1 and SOCS3, SOCS2 and CIS, SOCS5 and SOCS14 and SOCS9 and SOCS11) are most closely related (FIG. 4). Indeed, the SH2 domains of SOCS5 and SOCS14 are almost identical (FIG. 4B), and unlike CIS, SOCS1, SOCS2 and SOCS3, SOCS5 and SOCS14 have an extensive, though less well conserved, N-terminal region preceding their SH2 domains (FIG. 4A).

EXAMPLE 22 SOCS PROTEINS WITH WD-40 REPEATS

Four SOCS proteins with WD-40 repeats were identified. As with the SOCS proteins with SH2 domains, pairs of these proteins appeared to be closely related. Full length cDNAs of mouse SOCS4 and SOCS6 were isolated and shown to encode proteins containing eight WD-40 repeats N-terminal of the SOCS box (FIG. 4) and SOCS4 and SOCS6 share 65% amino acid similarity. SOCS15 was recognised as an open reading frame upon sequencing BACs from human chromosome 12p13 and the syntenic region of mouse chromosome 6 [Ansari-Lari et al, 1997]. In the human, chimp and mouse, SOCS15 is encoded by a gene with two coding exons that lies within a few hundred base pairs of the 3′ end of the triose phosphate isomerase (TPI) gene, but which is encoded on the opposite strand to TPI(9). In addition to a C-terminal SOCS box, the SOCS15 protein contains four WD-40 repeats. Interestingly, within the EST databases, there is a sequence of a nematode, an insect and a fish relative of SOCS15. SOCS15 appears most closely related to SOCS13.

EXAMPLE 23 SOCS PROTEINS WITH ANKYRIN REPEATS

Three SOCS proteins with ankyrin repeats were identified. Analysis of partial cDNAs of mouse SOCS7, SOCS10 and SOCS12 demonstrated the presence of multiple ankyrin repeats.

EXAMPLE 24 EXPRESSION PATTERN OF SOCS PROTEINS

The expression of mRNA from representative members of each class of SOCS proteins—SOCS1 and SOCS1 and SOCS5 from the SH2 domain group, SOCS6 from the WD-40

repeat group and SOCS7 from the ankyrin repeat group was examined. As shown above, SOCS1 mRNA is found in abundance in the thymus and at lower levels in other adult tissues.

Since transcription of the SOCS1 gene is induced by cytokines, the inventors sought to determine whether levels of SOCS5, SOCS6 and SOCS7 mRNA increased upon cytokine stimulation. In the livers of mice injected with IL-6, SOCS1 mRNA is detectable after 20 min and decreases to background levels within 2 hours. In contrast, the kinetics of SOCS5 mRNA expression are quite different, being only detectable 12 to 24 hours after IL-6 injection. SOCS6 mRNA appears to be expressed constitutively while SOCS7 mRNA was not detected in the liver either before injection of IL-6 or at any time after injection.

Expression of these genes was also examined after cytokine stimulation of the factor-dependent cell line FDCP-1 engineered to express bcl-w. Again, SOCS6 mRNA was expressed constitutively.

EXAMPLE 25 SOCS4

Mouse and human SOCS4 were recognized through searching EST databases using the SOCS box consensus (FIG. 13). Those ESTs derived from mouse and human SOCS4 cDNAs are tabulated below (Tables 4.1 and 4.2). Using sequence information derived from mouse ESTs several oligonucleotides were designed and used to screen, in the conventional manner, a mouse thymus cDNA library cloned into λ-bacteriophage. Two cDNAs encoding mouse SOCS4 were isolated and sequenced in their entirety (FIG. 5) and shown to overlap the mouse ESTs identified in the database (Table 4.1). These cDNAs include a region of 5′ untranslated region, the entire mouse SOCS4 coding region and a region of 3′ untranslated region (FIG. 7). Analysis of the sequence confirms that the SOCS4 cDNA encodes a SOCS Box at its C-terminus and a series of 8 WD-40 repeats before the SOCS Box (FIGS. 6 and 7). The relationship of the two sequence contigs of human SOCS4 (h4.1 and h4.2) to the experimentally determined mouse SOCS4 cDNA sequence is shown in FIG. 7. The nucleotide sequence of the two human contigs is listed in FIG. 8.

SEQ ID NO:13 and 14 represent the nucleotide sequence of murine SOCS4 and the corresponding amino acid sequence. SEQ ID NO:15 and 16 are SOCS4 cDNA human contigs h4.1 and h4.2, respectively.

EXAMPLE 26 SOCS5

Mouse and human SOCS5 were recognized through searching EST databases using the SOCS box consensus (FIG. 4). Those ESTs derived from mouse and human SOCS5 cDNAs are tabulated below (Tables 5.1 and 5.2). Using sequence information derived from mouse and human ESTs, several oligonucleotides were designed and used to screen, in the conventional manner, a mouse thymus cDNA library, a mouse genomic DNA library and a human thymus cDNA library cloned into λ-bacteriophage. A single genomic DNA clone (57-2) and (5-3-2) cDNA clone encoding mouse SOCS5 were isolated and sequenced in their entirety and shown to overlap with the mouse ESTs identified in the database (FIGS. 8 and 9A). The entire coding region, in addition to a region of 5′ and 3′ untranslated regions of mouse SOCS5 appears to be encoded on a single exon (FIG. 8). Analysis of the sequence (FIG. 9) confirms that SOCS5 genomic and cDNA clones encode a protein with a SOCS box at its C-terminus in addition to an SH2 domain (FIGS. 8 and 9B). The relationship of the human SOCS5 contig (h5.1; FIG. 10) derived from analysis of cDNA clone 5-94-2 and the human SOCS5 ESTs (Table 5.2) to the mouse SOCS5 DNA sequence is shown in FIG. 8. The nucleotide sequence and corresponding amino acid sequence of murine SOCS5 are shown in SEQ ID NO:17 and 18, respectively. The human SOCS5 nucleotide sequence is shown in SEQ ID NO:19.

EXAMPLE 27 SOCS6

Mouse and human SOCS6 were recognized through searching EST databases using the SOCS box consensus (FIG. 4). Those ESTs derived from mouse and human SOCS6 cDNAs are tabulated below (Tables 6.1 and 6.2). Using sequence information derived from mouse ESTs, several oligonucleotides were designed and use to screen, in the conventional manner, a mouse thymus cDNA library. Eight cDNA cones (6-1A, 6-2A, 6-5B, 6-4N, 6-18, 6-29, 6-3N, 6-5N) cDNA clone encoding mouse SOCS6 were isolated and sequenced in their entirety and shown to overlap with the mouse ESTs identified in the database (FIGS. 11 and 12A). Analysis of the sequence (FIG. 12) confirms that the mouse SOCS6 cDNA clones encode a protein with a SOCS box at its C-terminus in addition to a eight WD-40 repeats (FIGS. 11 and 12B). The relationship of the human SOCS-6 contigs (h6.1 and h6.2; FIG. 4) derived from analysis of human SOCS6 ESTs (Table 6.2) to the mouse SOCS6 DNA sequence is shown in FIG. 11. The nucleotide and corresponding amino acid sequences of murine SOCS6 are shown in SEQ ID NO:20 and 21, respectively. SOCS6 human contigs h6.1 and h6.2 are shown in SEQ ID NO:22 and 23, respectively.

EXAMPLE 28 SOCS7

Mouse and human SOCS7 were recognized through searching EST databases using the SOCS box consensus (FIG. 4). Those ESTs derived from mouse and human SOCS-7 cDNAs are tabulated below (Tables 7.1 and 7.2). Using sequence information derived for mouse ESTs, several oligonucleotides were designed and use to screen, in the conventional manner, a mouse thymus cDNA library. One cDNA clone (74-10A-11) cDNA clone encoding mouse SOCS7 was isolated and sequenced in its entirety and shown to overlap with the mouse ESTs identified in the database (FIGS. 14 and 15A). Analysis of the sequence (FIG. 15) suggests that mouse SOCS7 encodes a protein with a SOCS box at its C-terminus, in addition to several ankyrin repeats (FIGS. 14 and 15B). The relationship of the human SOCS7 contigs (h7.1 and h7.2; FIG. 16) derived from analysis of human SOCS7 ESTs (Table 7.2) to the mouse SOCS7 DNA sequence is shown in FIG. 14. The nucleotide and corresponding amino acid sequences of murine SOCS7 are shown in SEQ ID NO:24 and 25, respectively. The nucleotide sequence of SOCS7 human contigs h7.1 and h7.2 are shown in SEQ ID NO:26 and 27, respectively.

EXAMPLE 29 SOCS8

ESTs derived from mouse SOCS8 cDNAs are tabulated below (Table 8.1). As described for other members of the SOCS family, it is possible to isolate cDNAs for mouse SOCS8 using sequence information derived from mouse ESTs. The relationship of the ESTs to the predicted coding region of SOCS8 is shown in FIG. 17. With the nucleotide sequence obtained from the ESTs shown in FIG. 18A and the partial amino acid sequence of SOCS8 shown in FIG. 18B. The nucleotide sequence and corresponding amino acid sequences for murine SOCS8 are shown in SEQ ID NO:28 and 29, respectively.

EXAMPLE 30 SOCS9

Mouse and human SOCS-9 were recognized through searching EST databases using the SOCS box consensus (FIG. 4). Those ESTs derived from mouse and human SOCS9 cDNAs are tabulated below (Tables 9.1 and 9.2). The relationship of the mouse SOCS9 contigs derived from analysis of the mouse SOCS9 EST (Table 9.1) to the human SOCS-9 DNA contig (h9.1; FIG. 21) derived from analysis of human SOCS9 ESTs (Table 9.2) is shown in FIG. 20. Analysis of the sequence (FIG. 22) indicates that the human SOCS9 cDNA encodes a protein with a SOCS box at its C-terminus, in addition to an SH2 domain (FIG. 19). The nucleotide sequence of muring SOCS9 cDNA is shown in SEQ ID NO:30. The nucleotide sequence of human SOCS9 cDNA is shown in SEQ ID NO:31.

EXAMPLE 31 SOCS10

Mouse and human SOCS10 were recognized through searching EST databases using the SOCS box consensus (FIG. 4). Those ESTs derived from mouse and human SOCS10 cDNAs are tabulated below (Table 10.1 and 10.2). Using sequence information derived from mouse ESTs, several oligonucleotides were designed and use to screen, in the conventional manner, a mouse thymus cDNA library. Four cDNA clones (10-9, 10-12, 10-23 and 10-24) encoding mouse SOCS10 were isolated, sequenced in their entirety and shown to overlap with the mouse and human ESTs identified in the database (FIGS. 22 and 23). Analysis of the sequence (FIG. 23) indicates that the mouse SOCS10 cDNA clone is not full length but that it does encode a protein with a SOCS box at its C-terminus, in addition to several ankyrin repeats (FIG. 22). The relationship of the human SOCS10 contigs (h10.1 and h10.2; FIG. 24) derived from analysis of human SOCS10 ESTs (Table 10.2) to the mouse SOCS10 DNA sequence is shown in FIG. 22. Comparison of mouse cDNA clones and ESTs with human ESTs suggests that the 3′ untranslated regions of mouse and human SOCS10 differ significantly. The nucleotide sequence of murine SOCS10 is shown in SEQ ID NO:32 and the nucleotide sequence of SOCS10 human contigs h10.1 and h10.2 are shown in SEQ. ID. NO:33 and 34, respectively.

EXAMPLE 32 SOCSC11

Human SOCS11 were recognized through searching EST databases using the SOCS box consensus (FIG. 4). Those ESTs derived from human SOCS11 cDNAs are tabulated below (Table 11.1 and 11.2). The relationship of the human SOCS11 contigs (h11.1; FIG. 25A, B), derived from analysis ESTs (Table 11.2) to the predicted encoded protein, is shown in FIG. 26. Analysis of the sequence indicates that the human SOCS11 cDNA encodes a protein with a SOCS box at its C-terminus, in addition to an SH2 domain (FIG. 26 and 25B). The nucleotide sequence and corresponding amino acide sequence of human SOCS11 are represented in SEQ ID NO:34 and 36, respectively.

EXAMPLE 33 SICS12

Mouse and human SICS-12 were recognized through searching EST databases using the SOCS box consensus (FIG. 4). Those ESTs derived from mouse and human SOCS12 cDNAs are tabulated below (Tables 12.1 and 12.2). Using sequence information derived from mouse ESTs, several oligonucleotides were designed and use to screen, in the conventional manner, a mouse thymus cDNA library. Four cDNA clones (10-9, 10-12, 10-23 and 10-24) encoding mouse SOCS12 were isolated, sequenced in their entirety and shown to overlap with the mouse and human ESTs identified in the database (FIG. 27 and 28). Analysis of the sequence (FIG. 28 and 29) indicates that the SICS12 cDNA clone encodes a protein with a SOCS box at its C-terminus, in addition to several ankyrin repeats (FIG. 27). The relationship of the human SOCS12 contigs (h12.1 and h12.2; FIG. 29) derived from analysis of human SOCS12 ESTs (Table 12.2) to the mouse SOCS12 DNA sequence is shown in FIG. 27. Comparison of mouse cDNA clones and ESTs with human ESTs suggests that the 3′ untranslated regions of mouse and human SOCS12 differ significantly. The nucleotide sequence of SOCS12 is shown in SEQ ID NO:37. The nucleotide sequence of human SOCS12 and contigs h12.1 and h12.2 are shown in SEQ ID NO:38 and 39, respectively.

EXAMPLE 34 SICS13

Mouse and human SOCS-13 were recognized through searching EST databases using the SOCS box consensus (FIG. 4). Those ESTs derived from mouse and human SOCS13 cDNAs are tabulated below (Tables 13.1 and 13.2). Using sequence information derived from mouse ESTs, several oligonucleotides were designed and use to screen, in the conventional manner, a mouse thymus and a mouse embryo cDNA library. Three cDNA clones (62-1 , 62-6-7 and 62-14) encoding mouse SOCS13 were isolated, sequenced in their entirety and shown to overlap with the mouse ESTs identified in the database (FIGS. 30 and 31A). Analysis of the sequence (FIG. 31) indicates that the mouse SOCS13 cDNA encodes a protein with a SOCS box at its C-terminus, in addition to a potential WD-40 repeat (FIGS. 30 and 31B). The relationship of human SOCS13 contigs (h13.1 and h13.2; FIG. 32) derived from analysis of human SOCS13 ESTs (Table 13.2) to the mouse SOCS13 DNA sequence is shown in FIG. 30. The nucleotide sequence and corresponding amino acid sequence of murine SOCS13 and shown in SEQ ID NO:40 and 41, respectively. The nucleotide sequence of human SOCS13 and shown in SEQ ID NO:40 and 41, respectively. The nucleotide sequence of human SOCS13 contig h13.1 is shown in SEQ ID NO:42.

Mice lacking SOCS-1 are born and appear outwardly normal. However, they fail to thrive and within two to three weeks are less than half the size of their normal littermates. All SOCS^(−/−)mice die before weaning with profound fatty degeneration of the liver (FIG. 1). Consistent with the SOCS-1 expression pattern outlined above, significant deficiencies in haemopoietic populations, particularly lymphocytes (FIG. 2), are also evident. These experiments highlight the indispensable nature of SOCS-1action, suggesting that negative regulation of cytokine signaling by this protein is critical in maintaining homeostasis in the liver, as well as in the proper control of the production of specific blood cells.

Analysis of β-galactosidase activity in mice which one SOCS1 allele has been replaced with β-gal has revealed expression in most thymocytes, as well as in the spleen and bone marrow, where it appears to be restricted largely to lymphoid population.

Example 34A

To explore the physiological role of SOCS-1, the inventors generated mice lacking this gene. SOCS-1 d efficient (SOCS-1^(−/−)) mice are born at the expected Mendelian frequency, appear normal for the first week. Between 9 and 21 days of age the mice succumb to an illness characterised by fatty degeneration and monocytic infiltration of the liver, monocytic infiltration of the pancreas and heart and a severe lymphopaenia. While the molecular basis of this disease was unclear the most parsimonious hypothesis, given the role of SOCS proteins in negative regulation of signal transduction, is that the SOCS-1^(−/−) mice are hyper-responsive to a cytokine known to have toxic side effects. Strikingly, the phenotype of the SOCS1^(−/−) mice was similar to that described for neonatal mice injected with interferon gamma from birth.

SOCS-1 mRNA expression is induced by IFNα, IFNβ and IFNγ

In order to determine whether the cellular response to IFNα, IFNβ or IFNγ might be regulated by SOCS-1, the inventors examined whether these cytokines induces expression of SOCS-1 mRNA and whether expression of SOCS-1 inhibited the biological effect of interferon's. Northern blot analysis of mRNA from the fibroblast cell lines 2FTGH revealed that expression of SOCS-1, SOCS-2, SOCS-3 and CIS mRNA was low or undetectable in unstimulated cells. Within 15 to 30 minutes of stimulation by IFNα, IFNβor IFNγ SOCS1 and to a lesser extent SOCS-3 mRNA were detectable with expression peaking at about 60 minutes. Little or no expression of either SOCS-2 or CIS was observed in response to any of three interferons. A similar pattern of induction of SOCS mRNA was observed upon treatment of the J774 macrophage cell line with IFNγ and, in the liver, following intravenous injection of IFNγ into mice.

Expression of SOCS-1 and SOCS-3 but not SOCs2 or CIS inhibits IFNα, IFNβ or IFNγ signaling

2FTGH cells were transfected with expression vectors encoding FLAG-tagged versions of SOCS-1, SOCS-2, SOCS-3 and CIS and clones stably expressing these proteins were selected. The capacity of these lines to respond to INFα, IFNβ or IFNγ was compared with control 2FTGH cells. Cells were infected with virus and incubated with various concentrations of each IFN. Wild type cells and those expressing SOCS-2 and CIS were protected from the effects of virus infection by 300 IU/ml IFNα or IFNγ and 10 IU/ml IFNβ. In contrast, those expressing SOCS-1 SOCS-3 exhibited reduced sensitivity to the protective effects of all three forms of IFN. In the case of IFNβ SOCS-1 expressing 2FTGH cells were at least 300-fold less sensitive and SOCS-3 expressing 2FTGH cells were 10 to 30-fold less sensitive than unmanipulated counterparts. A similar hypo-responsiveness was observed when the capacity of IFNβ to suppress cell proliferation was assessed.

Disease in SOCS-1−/− mice is predated by cellular responses to IFNγ

Three lines of evidence suggested that the pathology observed in SOCS-1 deficient mice might results from a hyper-responsiveness to IFNγ. These were (i) the capacity of IFNγ to stimulate SOCS-1 expression, (ii) the ability of SOCS-1 to inhibit IFNγ signaling when constitutively expressed and (iii) the similarity of the phenotype of SOCS-1 deficient mice and mice injected IFNγ. the inventors therefore examined whether SOCS-1 mice showed evidence of an ongoing response to IFNγ either during the first week of life prior to overt development of disease or during disease onset and progression in the second and third weeks of life.

Phosphorylation of the IFNγRα chain by JAK1 and JAK2 and the consequent activation of STAT1 are key elements in IFNγ signal transduction. In the livers of new born, six-day old and 14-day old SOCS-1−/− but not SOCS-1+/− or wild type mice tyrosine phosphyorlation of the IFNγRα was readily detectable. Likewise, activated STAT1, as measured by EMSA, was also detected in SOCS-1^(−/−) but not littermates, at the three time points examined.

Given the evidence of IFNγ signaling in SOCS-1 −/− mice examined the liver for expression of class I and II MHC, iNOS and IRF-1 hallmarks of a biological response to IFNγ. At birth and after 6 and 14 days of life wild type and SOCS-1 +/− mice showed a low level of expression of class I MHC and little or no expression of class II MHC, iNOS and IRF-1. In contrast, expression of each of these proteins was elevated in the livers of SOCS-1 −/− mice at each time point examined. Class I and II MHC expression was also found to be elevated in many cells of the haemopoietic system, notably thymic and splenic T cells, as well as bone marrow and spenic B cells and moncytes. Expression of markers of a response to IFNγ in the liver and the maemopoietic system occurred before the onset of the overt signs of disease in SOCS-1 −/− mice suggesting that it was not a secondary effect of the pathology observed.

Lack of IFNg completely ameliorates disease in SOCS-1 −/− mice

In order to determine whether a response to IFNγ was the basis of disease development in SOBS-1−/− mice the inventors performed two experiments; (i) injection of mice with neutralizing anti-IFNγ antibody and (ii) generation of mice lacking functional SOCS-1 and IFNγ genes.

Litters¹ of mice born following the mating of SOCS-1−/+ mice were injected twice weekly from birth with either anti-IFNYγ monoclonal antibody or an isotype control antibody. SOCS-1−/− mice injected with control antibody developed disease with the same onset as similar unmanipulated SOCS-1 −/−. The disease observed in unmanipulated and control antibody injected SOCS-1 −/-− mice were also similar and was characterised by fatty degeneration of the liver, monocytic infiltration of several organs including the heart, liver, pancreas and skin, a generalized reduction in the size of the thymus and selective loss of pre-B and mature B cells.

EXAMPLE 35 SOCS14

Mouse and human SOCS-14 were recognized through searching EST databases using the SOCS box consensus (FIG. 4). Those ESTs derived from mouse and human SOCS14 cDNAs are tabulated below (Tables 14.1 and 14.2). Using sequence information derived from mouse and human ESTs, several oligonucleotides were designed and use to screen, in the conventional manner, a mouse thymus cDNA library, a mouse genomic DNA library and a human thymus cDNA library cloned into λ-bacteriophage. A single genomic DNA clone (57-2) and (5-3-2) cDNA clone encoding mouse SOCS14 were isolated and sequenced in their entirety and shown to overlap with the mouse ESTs identified in the database (FIGS. 33 and 34A). The entire coding region, in addition to a region of 5′ and 3′ untranslated regions, of mouse SOCS14 appears to be encoded on a single exon (FIG. 33). Analysis of the sequence (FIG. 34) confirms that SOCS14 genomic and cDNA clones encode a protein with a SOCS box at its C-terminus in addition to an SH2 domain (FIGS. 33 and 34B). The relationship of the human SOCS14 contig (h14.1) derived from analysis of cDNA clone 5-94-2 and the human SOCS14 ESTs contig (14.2) to the mouse SOCS14 DNA sequence is shown in FIG. 33.

The nucletotide sequence and corresponding amino acid sequence of murine SOCS14 are shown in SEQ ID NO:43 and 44, respectively.

EXAMPLE 36 SOCS15

Mouse and human SOCS15 were recognized through searching DNA databases using the SOCS box consensus (FIG. 4). Those ESTs derived from mouse and human SOCS15 cDNAs are tabulated below (Tables 15.1 and 15.2), as are a mouse and human BAC that contain the entire mouse and human SOCS-15 genes. Using sequence information derived from the ESTs and the BACs it is possible to predict the entire amino acid sequence of SOCS15 and as described for the other SOCS genes it is feasible to design specific olignoucleotide probes to allow cDNAs to be isolated. The relationship of the BACs to the ESTs is shown in FIG. 35 and the nucleotide and predicted amino acid sequence of the SOCS-15, derived from the mouse and human BACs is shown in FIGS. 36 and 37. The nucleotide sequence and corresponding amino acid sequence of murine SOCS15 are shown in SEQ ID NO:46 and 47, respectively. The nucleotide and corresponding amino acid sequence of human SOCS15 are shown in SEQ ID NO:48 and 49, respectively.

EXAMPLE 37 SOCS INTERACTION WITH JAK2 KINASE

These examples show interaction between SOCS and JAK2 kinase. Interaction is mediated via the SH2 domain of SOCS1, 2, 3 and CIS. The interaction resulted in inhibition of JAK2 kinase activity by SOCS1.

The following methods are employed:

Immunoprecipitation: Cos 6 cells were transiently transfected by electroporation and cultured for 48 hours. Cells were then lysed on ice in lysis buffer (50 mM Tris/HCL, pH 7.5, 150 mM NaCl, 1% v/v Triton-X-100, mM EDTA, 1 mM Naf, 1 mM Na₃VO₄) with the addition of complete protease inhibitors (Boehringer Mannheim), centrifuged at 4° C. (14,000×g, 10 min) and the supernatant retained for immunoprecipitation. JAK2 proteins were Immunoprecipitation using 5 μl anti-JAK2 antibody (UBI). Antigen-antibody complexes were recovered using protein A-Sepharose (30 μl of a 50% slurry).

Western blotting: immunoprecipitates were analysed by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) under reducing conditions. Protein was then electrophoretically transferred to nitrocellulose, blocked overnight in 10% w/v skim-milk and washed in PBS/0.1% v/v Tween-20 (Sigma) (wash buffer) prior to incubation with either anti-phosphotyrosine antibody (4G10) (1:5000, UBI), anti-FLAG antibody (1.6 μg/ml) or anti-JAK2 antibody (1:2000, UBI) diluted in wash biffer/1% w/v BSA for 2 hr. Nitrocellulose blots were washed and primary antibody detected with either peroxidase-conjugated sheep anti-rabbit immunoglobulin (1:5000, Silenus) or peroxidase-conjugated sheep anti-mouse immunoglobulin (1:5000, Silenus) diluted in wash buffer/1% w/v BSA. Blots were washed and antibody binding visualized using the enhanced chemiluminescence (ECL) system (Amersham, UK) according to the manufacturers' instructions.

In-vitro kinase assay: An in vitro kinase assay was performed to assess intrinsic JAK2 kinase catalytic activity. JAK2 protein were immunoprecipated as described, washed twice in kinase assay buffer (50 mM NaCl, 5 mM MgCl₂, 5 mM M Cl2, 1 mM NaF, 1 mM Na₃VO₄, 10 mM HEPES, pH 7.4) and suspended in an equal volume of kinase buffer containing 0.25 μCi/ml (γ−³² P)−ATP (30 min, room temperature. (excess (γ−³² P)−ATP was removed and the immunoprecipitates analysed by SDS-PAGE under reducing conditions. Gels were subjected to a mild alkaline hydrolysis by treatment with 1 M KOH (55° C., 2 hours) to remove phosphoserine and phosphothreonine. Radioactive bands were visualised with IMAGEQUANT software on a PhosphorImage system (Molecular Dynamics, Sunnyvale, Calif., USA).

EXAMPLE 38 MAKING SOCS-1 KNOCKOUT CONSTRUCTS

Diagrams of plasmid constructs and knockout constructs are shown in FIGS. 51-53. The genomic SOCS-1 clone 95-11-10 was digested with restriction enzymes BamH1 and EcoR1 to obtain a 3.6 Kb DNA fragment 3′ of the coding region (SOCS-1 exon), which was used as the 3′ arm in the SOCS-1 knockout vectors. The ends of this fragment were then blunted. This fragment was then ligated into the following vectors:

pBagalpAloxNeo and pBgalpAloxNeoTK which had been lineraized at the unique Xho1 site and then blunted. This ligation resulted in the formation of the following vectors:

3′SOCS-1 arm in pBgalpAloxNeo and 3′SOCS-1 arm in pBgalpAloxNeoTK

The 5′arm of the SOCS-1 knockout vectors was constructed by using PCR to generate a 2.5 Kb PCR product from the genomic SOCS-1clone 95-11-10 just 5′ of the SOCS-1 coding region (SOCS-1 exon). The oligo's used to generate this product were:

5′ oligo (sense) (2465)

AGCT AGA TCT GGA CCC TAC AAT GGC AGC [SEQ ID NO:49]

3′ oligo (antisense) (2466)

AGCT AG ATC TGC CAT CCT ACT CGA GGG GCC AGC TGG [SEQ ID NO:50]

The PCR product was then digested with the restriction enzyme BglII, to generate BglII ends to the PCR product. This 5′ SOCS-1 PCR product, with BglII, ends was then ligated as follows: 3′SOCS-1 arm in pBgalpAloxNeo and 3′SOCS-1 arm in pBgalpAloxNeoTK, which had been linerized with the unique restriction enzyme BamH1. This resulted in the following vectors being formed:

5′&3′SOCS-1 arms in pBgalpAloxNeo and 5′&3′SOCS-1 arms in pBgalpAloxNeoTK

These were the final SOCS-1 knockout constructs. Both these contructs lacked the entire SOCS-1 coding region (SOCS-1 EXON), being replaced with portion of the Bgal, B globin polyA, PGK promoter, neomycin and PGK polyA sequences. The 5′'&′SOCS-1 arms in pBgalpAloxNeoTK vector also contained the tymidine kinase gene sequence, between the neomycin and PGK poly A sequences.

The vectors: 5′&3′SOCS-1 arms in pBgalpAloxNeo and 5′&3′SOCS-1 arms in pBgalpAloxNeoTK were linerized with unique restriction enzyme Not1 and then transfected into Embroyonic stem cells by electroporation. Clones which were resistant to neomycin were selected and analysed by southern blot to determine if they contained the correctly integrated SOCS-1 targeting sequence. In order to determine if correct integration had occured, genomic DNA from the neomycin resistant clones was digested with the restriction enzyme EcoR1. The digested DNA was then blotted onto nylon filters and probed with a 1.5b EcoR1/Hind III DNA fragment, which was further 5′ of the 5′ arm sequence used in the knockout constructs. The band sizes expected for correct integration were:

Wild type SOCS-1 allelle 5.4 Kb

SOCS-1 knockout allele 8.2 Kb in 5′&3′ SOCS-1 arms in pBgalpAloxNeo or 11 kb in 5′&3′SOCS-1 arms in pBgalpAloxNeoTK transformed cells.

EXAMPLE 39 ANALYSIS OF SOCS-1 DELETION MUTANTS

SOCS-1 deletion mutants were generated by PCR to give fragments with Asc 1Mlu 1 linkers at the N- and C-terminus and subcloned into PEF-FLAG-I (found at http://www.wehi.edu.au/willson vectors) to give N-terminal Flagged proteins. SOCS-1 deletion mutants were therefore constructed in which the N-terminal domain was deleted, retaining amino acid 77-211 (ΔN), the C-terminal domain encompassing the SOCS box was deleted, retaining amino acids 1-169 (ΔC) and both the N-terminal region and the SOCS box were deleted, retaining amino acids 77-169, leaving an intact SH2 domain (ΔN/C). In addition, a aconstruct was created in which the SH2 domain and the SOCS box were both deleted, retaining the N-terminal 81 amino acids (ΔSH2/C). These constructs were transfected into parental M1 cells and with the exception of ΔSH2/C, several stable transfectants were obtained for each construct. Protein expression of the deletion mutants was confirmed by immunoprecipitation and Western blot analysis using anti-Flag antibodies.

These constructs were then assessed for their ability to inhibit IL-6 and LIF signalling in several different assay systems.

Colony assays is soft agar

Cultures were performed as previously described (Metcalf, 1984). M1 parental cells form large compact colonies soft agar. When cells are incubated in the presence of IL-6 or LIF the colonies are dispersed with a halo of cells migrating out from the central core. At high concentrations of cytokine, the number of colonies observed is highly diminished, a phenomenon known as clonal suppression. M1 cells which constitutively express SOC-1 are unable to respond to either IL-6 or LIF, as both macrophage differentiation and clonal suppression are inhibited (Starr et al. 1997).

To assess the ability of the SOCS-1 deletion mutants to suppress M1 cell differentiation, cells expressing the various constructs were plated in agar in the presence or absence of increasing concentration of IL-6 or LIF. M1 cells expressing either SOCS-1 ΔN or ΔN/C protein at eqivalent levels to cells expressing full-length SOCS-1 were unable to block IL-6 or LIF-induced differentiation, responding to growth factor in a similar manner to the parental M1 cells (M1-P). These results indicated that the N-terminal region of SOCS-1 was critical for inhibition of N1 macrophage differentiation.

2. Inhibition of LIF-induced luciferase activity in 293T cells

293T is a human fibroblast line expressing endogenous LIF receptors. Briefly, 293T cells were plated into either 24well plates at 1×105 cells/well or 10 cm dishes at 2×106 cells/dish. The LIF responsive promoter-luciferase gene (APRE-luc) and has been described previously (Mashsuhara et al. 1997). The positive control vector Sra-b-gal encoding the β-gel galactosidse gene has also been described Ogilvy et al. 1998). Plasmids of reporter genes with either vector alone or pEF-FLAG SOCS constructs were introduced into cells using FuGENE transfection reagent (Boehringer Mannheim) according to the Manufacturers' instructions and harvested after 48 hr. Cells were stimulated with or without 10 ng/ml hLIF overnight prior to lysis with 40 ml Reporter Lysis Buffer (Promega) containing protease inhibitors. Lysates were then assayed for luciferase and β-galactosidase activity.

293T cells were transiently transfected with the LIF-responsive reporter construct, APRE-luc and LIF induction of luciferase activity measured. A clear increase in luciferase activity is observed via JAK activation of Stat3 which dimerizes and in turn binds to the APRE (acute phase response element). The differential ability of the various SOCS proteins to modulate IL-6 and LIF signalling in M1 cells was confirmed by transient expression of the APRE-luc reporter gene with or without Flag-tagged SOCS-1, SOCS-2, SOCS-3, CIS, SOCS5 and SOC-6 in 293T cells. SOCS-1 and SOCS-3 completely abolished the LIF-induced activation of luciferase activity, whereas SOCS-2, CIS and SOC-6 had no effect, and SOCS-5 partially inhibited the LIF response. Transfection efficency was controlled for by co-expression of a b-galactosidase reporter construct under a constitutive promoter (Sra-b-gal) and luciferase activity normalized against the b-galactosidase results. Similarly, the ability of the SOCS-1 deletion mutants to inhibit M1 differentiation was paralleled by their ability to inhibit luciferase activity when transiently expressed in 293T cells. In addition, expression of SOCS1 ΔSH2C did not inhibit LIF-induced luciferase activity, suggesting that the N-terminal region alone was insufficient to mediate SOCS-1 inhibition of LIF signalling.

3. Inhibition of JAK2 autophosphorylation in an in vitro kinase assay

To further investigate the function of the SOCS-1 deletion mutants, the inventors examined the ability of the different SOCS proteins to directly inhibit JAK kinase activity. The methods used are outlined below.

Briefly, cell lysates were prepared as previously described (Nicholson et al. 1995). Proteins were immunoprecipitaed with either anti-JAK2 antibodies (UBI) or anto-Flag antibody conjugated to Sepharose (M2;Eastman Kodak Company) and proteins separated on 4-15% w/v gradient SDS-PAGE gels. Protein was then electrophoretically transferred to PVDF membranes. Membranes were blocked overnight in 10% w/v skim milk and incubated with primary antibody for 2 hr. Antibody binding was visualized with either peroxidase-conjugated anti-rabbit Ig (Silenus) or peroxidase conjugated anti-mouse IgFc, which specifically recognizes the immunoglobulin heavy chain (Jackson Laboratories) and the enhanced chemiluminescence (ECL) system (Amersham). Anti-JAK2 immunoprecipitates were washed and incubated with g-ATP as previously described (nicholson et al. 1995).

Flag-tagged JAK2 was transiently expressed in Cos cells with or without the various SOCS-1 deletion mutants. Cells were lysed and JAK2 proteins immunoprecipitation, incubated in kinase buffer containing radiolabelled γ-ATP, and the proteins seperated by SDS-PAGE gel (as described above). Incorportion of radiolabelled phosphate into the JAK2 protein (autophosphorylation) was then visualised using a phosphoimager. Co-expression of full length SOCS-1 dramatically inhibited JAK2 autophosphorylation. Three of the SOC-1 deletion mutants were tested (ΔC, □N/C, ΔN) for their ability to functionally inhibit the JAK2 kinase a ctivity. Co-expression of each of these constructs with JAK2 indicated that they were also able to inhibit JAK2 kinase activity to the same degree as full-length SOCS-1, Immunoprecipitation of equal amounts of JAK2 protein was demonstrated by Western blot with anti-Flag antibodies. Expression levels of the various SOCS-1 deletion mutants was determined by immunoprecipitation and Western blot with anti-Flag antibodies. These results indicate that at least in an over-expression system where JAK2 is constitutively active (ie. not a ligand-inducible system), the SH2 domain of SOCS-1 is sufficient to inhibit JAK kinase assay.

JAK2 was transiently expressed in Cos cells with or without the various SOCS-1 deletion mutants. Cells were lysed and JAK2 tyrosine phosphorylation assessed by Western blot with anti-phosphotyrosine antibodies. Inhibition of JAK2 tyrosine phosphorylation correlated with the ability of the various SOCS-1 deletion mutants to inhibit JAK2 kinase activity. In addition, all three of the SOCS-1 mutants tested (ΔC, ΔN/C, ΔN) appeared able to associate with JAK2.

The data obtained with the SOCS-1 deletion mutants indicate that the N-terminal region of SOCS-1 was required for its ability to inhibit LIF and IL-6 signaling, particularly with respect to induction of M1 cell differentiation. In addition, the in vitro kinase data indicates that inhibition of JAK kinase activity is mediated through the SOCS-1 SH2 domain

EXAMPLE 40 SOCS CHIMAERAS

In order to further investigate the importance of the difference SOCS domains, a series of chimeric proteins were created in which the N-terminal domain of SOCS-1 was replaced with the either the N-terminal domain of SOCS-2, SOCS-3, SOCVS-4, SOCS-5 pr SOCS-6. A series of chimeric proteins were also created in which the SH2 domain of SOCS-1 was replaced with either the SH2 domain of SOCS-2, SOCS-3, CIS , SOCS-5 or SOCS-6.

To facilitate the synthesis of chimeric mouse SOCS1 cDNAs on Xho I site was introduced at the boundary between the N-terminal region and SH2 domain of the SOCS1 cDNA (see FIG. 42). Two nucleotide changes were introduced using a PCR-based technique known as splicing by overlap extension (Horton et al. 1989). The PCR fragment, designated mSOCS1mutXho, was then cloned into the Kpn I and Sac I sites of pBLUESCRIPT SKII(+) [Stratagene]. In order to facilitate cloning of the DNA fragments into the mammalian expression vector pEF-FLAG-I (found at http://www.wehi.edu.au./willson vectors), an in-frame Asc I restriction enzyme site was introduced one amino acid after the predicted translational start site and Mlu I site was inserted immediately before the stop codon of the mouse SOCS1 cDNA. Since the C<G nucleotide alteration leasts to a Δ<E amino acid substitution at position 76the SOCS1mutXho cDNA was cloned into the Mlu I site of pER-FLAG-I and shown to have similar activity to wild-type SOCS-1 in the luciferase assay (FIG 42).

Hybrid cDNAs, in which the N-terminal region or the SH2 domain of the SOCS1 sequence were replaced with homologous regions of mouse CIS, SOCS2, SOCS3, SOCS5 or SOBS6, were synthesized from PCR generated restriction fragments (N-terminal regions were cloned in ASc I-XHho I fragments and SH2 domain fragments were clone in as Xho I-Not I fragments). All the hybrid cDNAs were then cloned in to the Mlu I site of pEF-FLAG-I in order to express mouse SOCS1 domain swap mutant proteins with an N-terminal FLAG epitope tag. Constructs were sequenced in their entirety before use. The exact specification of sequences present in each SOCS1 domain swap mutant is listed in Table 16.

TABLE 16 CONSTRUCTION OF MOUSE SOCS1 DOMAIN SWAP MUTANT PROTEINS Hybrid Amino Acid Sequence Specifications SOCS1-CNT CIS(2-80):SOCS1(75-212) SOCS1-2NT SOCS2(2-46):SOCS1(75-212) SOCS1-3NT SOCS3(2-44):SOCS1(75-212) SOCS1-SNT SOCSS(2-379):SOCS1(75-212) SOCS1-6(9)NT SOCS6(2-380):SOCS1(75-212) SOCS1-CSH2 SOCS1(2-78):CIS(81-218):SOCS1(172-212) SOCS1-2SH2 SOCS1(2-78):SOCS2(47-159):SOCS1(172-212) SOCS1-3SH2 SOCS1(2-78):SOCS3(4S-185):SOCS1(172-212) SOCS1-5SH2 SOCS1(2-78):SOCS5(380-480):SOCS1(172-212) SOCS1-6(9)SH2 SOCS1(2-78):SOCS6(381-494):SOCS1(172-212)

Amino acid sequences are designated according to the following example. CIS(2-80):SOCS1(75-212) (hybrid SOCS1-CNT) denotes that amino acid residues 2 to 80 are derived from N-terminal region of the mCIS sequence and amino acid residues 75 to 212 are derived from mouse SOCS1 sequence.

These chimeric constructs were Flag-tagged, transiently expressed in 293T cells and LIF-induction of luciferase activity assayed. In contract to wild-type SOCS-1, none of the chimeric proteins were able to inhibit LIF-induction of luciferase activity. Therefore, the N-terminal region of SOCS-1 cannot be functionally replaced by any of the SOCS-2, SOCS-3, CIS, SOCS-4, SOCS-5 and SOCS-6 N-terminal domains. Likewise, Although none of the introduced SH2 domains is able to fully replace the SOCS-1-SH2, partial inhibition of LIF-induced luciferase activity was observed with chimeric SOCs proteins, SOCS-1-3SH2, SOCS-1CSH2, and SOCS-1-5-SH2.

The data in both M1 cells and 293T cells indicated that whilst the N-terminal region was critical SOCS-1 function, the SH2 domains of several SOCS proteins was sufficient for some, though not normal, level of activity.

Previous work has shown that mutation of a conserved arginine residue to lysine within SH2 domains results in a non-functional domain. Mouse SOCS1, SOCS2, SOCS3 and CIS cDNAs, in which an R>K amino acid substitution in the SH2 domain was introduced (see FIG. 42), were generated using the PCR-based technique, splicing by overlap extension. To facilitate cloning of the PCR-generated fragments into pEF-FLAG-I, an in-frame Asc I restriction enzyme site was introduced immediately after the predicted translation start site and an Mlu I site was inserted immediately before the translation stop site. Expression construct pEF-FLAG-I/mSOCS1-R10K encodes a mSOCS1 protein with an R>K amino acid substitution at position 105 in the SH2 domain. pEF-FLAG-I/mSOCS2-R73K, pEF-FLAG-I/mSOCS3-R17K, pEF-FLAG-I/mCIS-R107K encode mSOCS2, SOCS3 and CIS proteins with R>K amino acid substitutions at the equivalent position in the SH2 domain, respectively.

To further confirm that the SH2 domain of SOCS-1 was required for activity, point mutations were made in each of the SOCS protein SH2 domains (SOCS-1 to SOCS-3, CIS; FIG. 42) changing the conserved arginine residue to a lysine. These constructs were then transiently expressed in the 293T reporter gene system. SOCS-1 containing a non-functional SH2 domain (SOCS-1-R105K) was unable to inhibit LIF-induced luciferase activity, providing further evidence that the SH2 domain has a critical role in SOCS-1 function. Further, a mutation of the SOCS-3-SH2 domain (SOCS-3-R71K) did not abrogate the ability of SOCS-1 to inhibit LIF signalling. This not only confirms a critical role for the SOCS-1-SH2 domain in LIF signalling, but is the first evidence to suggest that although both SOCS-1 and SOCS-3 are able to inhibit LIF and IL-6, signal transduction, they may do so through entirely different mechanisms.

EXAMPLE 41 Biochemical Analysis of SOCS Action

The inventors sought further evidence as to the molecular site of action of the various SOCS proteins.

M1 parental cells (M1-P) and M1 cells constitutively expressing a Flag-tagged SOCS-1 protein were serum-starved for 2 hrs and stimulated for 0, 5, 10 minutes with 104 U/ml mLIF. Cells were lysed and JAK proteins immunoprecipitated with 5 ml anti-JAK1 antibodies (UBI) and protein-A-Sepharose. Precipitates were washed and proteins separated by SDS-PAGE on a 4-15% w/v gradient gel, prior to anlaysis by Western blot with anti-phosphotyrosine antibodies. In parental M1 cells JAK1 was clearly phosphorylated in response to LIF. In contrast, constitutive expression of SOCS-1 inhibited JAK1 tyrosine phosphorylation.

High level expression of JAK2 protein in Cos cells results in a constitutively active JAK protein, presumably due to dimerisation and cross-phosphorylation. Flag-tagged JAK2 was, therefore, transiently expressed by electroporation in Cos cells with and without co-expression of Flag-tagged SOCS-1. After 48 hours cells were lysed on ice and JAK2 proteins immunoprecipitated using 5 ml anti-JAK2 antibody (UBI). Immunoprecipitates were washed divided in two and half the proteins subjected to an in vitro kinase assay as previously described. Proteins were then separated by SDS-PAGE on a 4-15% w/v gradient gel and the gel treated with KOH to remove phosphoserine and phosphothreonine. Incorporation of radiolabelled phosphate was detected using a phosphoimager. The remaining half of the immunoprecipitation was run on a 4-15% w/v gradient gel and analysed by Western blot with anti-JAK2 antibodies (UBI) to demonstrate equal immunoprecipitation.

Lysates from Cos cells expressing either JAK2 or both JAK2 and SOCS-1, were run on a 4-15% w/v gel, electrophoretically transferred to PVDF and analysed with anti-phosphotyrosine antibodies at a 1:5000 dilution (4G10;UBI). A single phosphorylated band corresponding to JAK2 was observed in parental M1 cells, which was not evident in cells expressing SOCS-1. Lysates were re-probed with anti-Flag antibodies to demonstrate equal loading of JAK2 protein. Expression of SOCS-1 was therefore able to inhibit both JAK2 kinase activity or autophosphorylation and tyrosine phosphorylation.

The ability of SOCS family members, SOCS-2, SOCS-3 and CIS to inhibit JAK2 kinase activity was further investigated. Flag-tagged JAK2 was transiently expressed by electroporation, with or without, Flag-tagged-SOCS-1, SOCS-2, SOCS-3 and CIS in Cos cells. After 48 hours cells were lysed on ice and JAK2 proteins immunoprecipitated using 5 ml and anti-JAK2 antibody (UBI). Immunoprecipitates were washed and subjected to an in vitro kinase assay as previously described. Proteins were then separately by SDS-PAGE on a 4-15% w/v gradient gel and the gel treated with KOH to remove phosphoserine and phosphothreonine. Incorporation of radiolabelled phosphate was detected using a phosphoimager (FIG. 62b). As had been demonstrated previously, co-expression of SOCS-1 inhibited JAK2 kinase activity. Co-expression of SOCS-2 and CIS also appeared to inhibit JAK2 kinase activity, whilst co-expression of SOC-3 did not inhibit kinase activity. These results suggests that SOCS-1 and SOCS-3 may have a differential ability to inhibit JAK2 kinase activity.

M1 cell lines stably expressing Flag-tagged SOCS-1, SOCS-2, SOCS-3 or CIS proteins, were lysed and the SOCS proteins immunoprecipitated using anti-Flag antibodies conjugated to sepharose. Immunoprecipitates were washed and the proteins were separated by SDS-PAGE on a 4-15% w/v gradient gel. Proteins were then electrophoretically transferred to PVDF membrane and analysed using anti-Flag antibodies. Expression levels of SOCS-2 (S2) were considerably higher than the other SOCS proteins with the expression levels of SOCS-1 (S1) being the lowest.

Stat3 tyrosine phosphorylation has previously been implicated in IL-6-induced differentiation, both by the use of dominant negative Stat3 constructs and by specific tyrosine mutations within the IL-6 singalling chain, gp130, which block recruitment Stat3 to the receptor complex. The inventors examined, therefore, LIF-induced Stat3 tyrosine phosphorylation in M1 cells expressing the various SOCS-1, SOCS-2, SOCS-3 and CIS. M1 cell lines stably expressing the various SOC proteins, were serum-starved for 1.5 hours prior to stimulation with 104 U/ml mLIF for 0, 5 and 10 minutes. Cells were then lysed, Stat3 proteins immunoprecipitated and analyzed by Western blot with antibodies specific to phosphorylated Stat3 (BioLabs). Stat3 was rapidly tyrosine phosphorylated in parental M1 cells in response to LIF. In contrast, Stat3 tyrosine phosphorylation was inhibited in M1 cells expressing SOCS-1 and SOCS-3. In each instance, Stat3 tyrosine phosphorylation correlated inversely with the ability of the expressed SOCS protein to inhibit M1 cell differentiation. This indicates that Stat3 has a critical role in IL-6 induced M1 cell differentiation and further suggests that the ability of SOCS-1 and SOCS-3 to inhibit M1 differentiation may be mediated through inhibition of the JAK-STAT pathway. Western blots were stripped and re-probed with anti-Stat3 antibodies to demonstrate equal loading of Stat3 protein.

EXAMPLE 42 Expression of SOCS-2, SOCS-3 and CIS in M1 Cells

cDNAs encoding epitope-tagged SOCS-2, SOCS-3 or CIS were generated by subcloning the entire coding region of each gene into the pEF-BOS expression vector, engineered to encode an in-frame FLAG epitope downstream of an initiation methionine. Using electroporation, M1 cells were transfected with 20 mg of linearised expression plasmid and 2 mg of a linearised plasmid in which transcription of a cDNA encoding puromycin N-acetyl transferase was driven from the mouse phosphoglycerokinase promoter. After 48 hours in culture, transfected cells were selected with 20 μg/ml puromycin. Puromycin-resistant cells were screened for the expression of SOCS-2, SOCS-3 or CIS by immunoprecipitation and Western blotting of cell extractors with the M2 anti-FLAG monoclonal antibody.

In order to assay the differentiation of M1 cells in response to cytokines, 300 cells were cultured in 35 mm Petri dishes containing 1 ml of DME supplemented with 20% v/v foetal calf serum (FCS), 0.3% w/v agar and0.1 ml of serial dilutions of interleukin 6 (IL-6). After 7 days culture at 37° C. in a fully humidified atmosphere, containing 10% v/v CO₂ in air, colonies of M1 cells were counted and classified as differentiated if they were composed of dispersed cells or had a corona of dispersed cells around a tightly packed centre. The total number of colonies in each dish were counted to determine the degree of clonal suppression induced by IL-6.

M1 cells expressing SOCS-2 were slightly hyporesponsive to differentiation induced by IL-6. However, IL-6 was unable to induce clonal suppression in these cells. The level of SOCS-2 expression in M1 cells was 10-fold higher than that for the other SOCS proteins. SOCS-3 expression in M1 cells completely inhibited the ability of IL-6 to induce either clonal suppression or differentiation in agar in response to IL-6, similar to the action of SOCS-1. M1 cells expressing CIS responded to IL-6 in a similar manner to parental M1 cells.

EXAMPLE 43 Knockout of SOCS Genes

In vitro studies have clearly identified the SOCS protein as key negative regulators of signal transduction. Moreover, injection of cytokines into mice has been shown to result in increased transcription of SOCS genes, implicating these proteins in regulation of cytokine responses in vivo. In order to determine the physiological processes regulated by each SOCS gene the inventors carry out experiments to “knockout” individual SOCS genes in mice. The first step in doing this is to clone genomic DNA encoding each of the SOCS genes. The maps of the genes for mouse SOCS-1, SOCS-2, SOCS-3, SOCS-5, SOCS-9 and SOCS-11 are shown in FIGS. 43 A-F).

Generation and analysis of SOCS-1 knockout mice

To construct the SOCS1 targeting vector, a 5′ arm extending approximately 2.5 kb from the protein initiation ATG was generated by PCR using specific SOCS1 oligonucleotides and genomic clone pgmSOCS1 95-11-10 as template. This fragment was fused to the ATG of β-galactosidse via the BamHI site in the plasmid vector pβgalpAloxneo (FIG. 38). The 3′ arm, a 3.2 kb BamHI-EcoRV fragment from pgmSOCS1 95-11-10 (FIG. 44) was blunted and ligated into the XhoI (blunted) site of pβgalpAloxneo that already contained the 5′ arm. This targeting vector was linearised with NotI and electroporated into W9.5 embryonic stem cells. After 48 hours, transfected cells were selected in 175 μg/ml G418 and resistant clones picked and expanded after a further 8 days. Clones in which the targeting vector had recombined with the endogenous SOCS1 gene were identified by hybridising EcoRI-digested genoic DNA with 1.5 kb EcoRI-HindIII fragment from pgmSOCS1 95-11-10. This probe (probe A, FIG. 44), which is located 5′ to the SOCS1 sequences in the targeting vector, distinguished between the endogenous (5.3 kb) and targeted (8.0 kb) SOCS1 loci (FIG. 44). The appropriate homologous recombination event was confirmed in ES clones with probe B, a 0.7 kb BamHI-NheI fragment from pgmSOCS1 95-11-10 (FIG. 44) situated 3′ to the SOCS1 gene. Genomic DNA was digested with EcoRI for 16 hrs at 37° C., electrophoresed through 0.8% w/v agarose, transferred to nylon membranes and hybridised to ³²P-labelled probe in a solution containing 0.5M sodium phosphate, 7% w/v SDS, 1 mM EDTA and washed in a solution containing 40 mM sodium phosphate, 1% w/v SDS at 65° C. Hybridising bands were visualised by autoradiography for 16 hours at −70° C. using Kodak XAR-5 film and intensifying screens. A targeted ED cell clones, W9.5SOCS1 1.4-A8 was injected into C57B1/6 blastocysts to generate chimeric mice. Male chimeras were mated with C57B1/6 females to yield SOCS1 heterozygotes which are currently being interbred to produce wild-type (SOCS-1+/+), heterozygous (SOCS +/−) and mutant (SOCS −/−) mice. The genotypes of offspring were determined by Southern Blot analysis of genomic DNA extracted from tail biopsies.

Mice lacking expression of SOCS-1 have been generated by replacing the entire coding region of SOCS-1 with bgal. In both heterozygous mice (SOCS-1+/−) and homozygous knockout mice (SOCS-1−/−), bgal expression should accurately reflect the normal tissue expression of SOCS-1. This experiment was a trial to determine whether βgal expression could be detected in SOCS-1+/− mice. The thymus was specifically chosen from anlaysis as Northern blots have previously shown high levels of constitutive SOCS-1 expression in this tissue. The procedure for FACSgal involves loading cells with a bgal substrate, fluorescein di-β-D-galactopyranoside (FDG), and allowing time for the bgal to convert this substrate to fluorescein, which can be detected by FACS analysis.

The steps in this procedure are:

1) Tissues (thymus, spleen femur harvested from:

a) SOCS-1+/−mice

b) SOCS-1+/+littermates (negative control)

c) ROSA mice (positive control)

2) Single cell suspension of each tissue obtained by flushing through KDS buffer containing 5% v/v FCS.

3) RBC lysis.

4) Cell pellet resuspended in 150 ml KDS/5% v/v FCS.

5) Hypotonic loading: Warmed cells diluted 1:1 with 2 mM FDG. Incubated at 37° C. for 120 secs.

6) Cells incubated on ice for ˜3 hr to allow hydrolysis of FDG to fluorescein. 1 mg/ml propidium iodide added to cells prior to FACSCAN analysis.

From this analysis we have shown bgal expression is high (92% of cells) in the thymus of SOCS-1+/−mice, as expected. A smaller percentage of cells from spleen (12%) and bone marrow (22%) were also expressing βgal. The high level expression of SOCS-1 in the thymus accurately reflects the expression of SOCS-1 observed by Northern anlaysis showing that bgal expression in SOCS-1+/−mice can be used as a marker for SOCS-1 expression. Moreover, since β-galactosidase expression, and indeed any other marker in which is inserted into the SOCS-1 locus, like green flourescent protein, will be transcribed in response to cytokines these mice are extremely useful reagents for monitoring responses to cytokines in vivo, in addition to being bred to yield mice which lack SOCS-1.

Generation of SOCS2 knockout mice:

To construct the SOCS2 targeting vector, a 5′ arm extending approximately 2.0 kb from the protein initiation ATG was generated by PCR using specific SOCS2 oligonucleotides and genomic clone pgmSOCS2 57-60-1-45 as template. This fragment was fused to the ATG of β-galactosidase via the BamHI site in the plasmid vector pβgalpAloxneo (FIG. 38). The 3′ arm, a 3.7 kb EcoRI fragment from pgmSOCS2 57-60-1-45 (FIG. 45) was blunted and ligated into the XhoI (blunted) site of pβgalpAloxneo that already contained the 5′ arm. This targeting vector was linearised with NotI and electroporated into BRUCE 4 embryonic stem cells. Transfected cells were selected in G418 and resistant clones picked and expanded. Clones in which the targeting vector had recombined with the endogenous SOCS2 gene were identified by hybridising EcoRV-digested genomic DNA with a 1.8 kb EcoRI-EcoRV fragment from pgmSOCS2 57-60-1-45. This probe (probe A, FIG. 45), which is located 3′ to the SOCS2 sequence in the targeting vector, distinguished between the endogenous (greater than 14 kb) and targeted (7.5 kb) SOCS2 loci (FIG. 45). Several targeted ES cell clones have been identified and are currently being injected into blastocysts to generate chimeric mice.

Generation of SOCS3 knockout mice:

To construct the SOCS3 targeting vector, a 5′ arm extending approximately 3.0 kb from the protein initiation ATG was generated by PCR using specific SOCS3 oligonucleotides and genomic clone pgmSOCS3 95-3 Xba as template. This fragment was fused to the ATG of β-galactosidase via the BamHI site in the plasmid vector pβgalpAloxneo. The 3′ arm is a 4.2 kb XbaI-XhoI fragment from pgmSOCS3 95-3 (FIG. 46). Initially, a 7.4 kb XbaI-HindII fragment from this genomic clone was ligated into pBluescript, from which the 3′ arm was excised as a XhoI fragment and ligated into the XhoI site of pβgalpAloxneo that already contained the 5′ arm. This targeting vector was linearised with AscI and is currently being electroporated into BRUCE 4 embryonic stem cells.

Generation of CIS knockout mice:

To construct the CIS targeting vector, a 5′ arm extending approximately 1.5 kb from the protein initiation ART was generated by PCR using specific CIS oligonucleotides and genomic clone pgmCIS 57-7-1-26 as template. This fragment was fused to the ATG of β-galactosidase via the BamHI site in the plasmid vector pβgalpAloxneo (see FIG. 38) into which the 3′ arm, a 3.2 kb BamHI fragment from pgmCIS 57-7-1-26 had already been inserted. The 3+ arm fragment was blunted and ligated into the XhoI (blunted) site of pβgalpAloxneo. The final targeting vector was linearised with NotI and electroporated into BRUCE 4 embryonic stem cells. After selection in G418, resistant clones were picked and expanded. Clones in which the targeting vector have recombined with the endogenous CIS gene are currently being identified by hybridising EcoRI-digested genomic DNA with a 0.8 kb BamHI-Ndel fragment from pgmCIS 57-7-1-26. This probe (probe A, FIG. 47), which is located 3′ to the CIS sequences in the targeting vector, will distinguish between the endogenous (10 kb) and targeted (8 kb) CIS loci (FIG. 47).

EXAMPLE 44 SOCS-1 Fusion Proteins with Green Fluorescent Protein

The inventors consider that if the SOCS-1 protein is active as a fusion protein with an easily visualized marker, then this would be a valuable reagent for both monitoring expression and intracellular location of SOCS-1, as well as an extremely useful reagent for monitoring a cells response to cytokines (since production of SOCS-1 is tightly regulated by cytokine). In order to test whether SOCS-1 is active as a fusion protein, the inventors have made certain vectors. Briefly, using the PCR, a derivative of the mouse SOCS1 cDNA was generated that encoded an N-terminal GFP tag (MARQSKGEELFT . . . ELYKTR [SEQ ID NO:51]) preceding the coding region (minus ATG) of mSOCS1 (see FIG. 48A), designated pEF-GFP-I/mSOCS1. Details of mammalian expression vector pEF-GFP-I can be found at http://www.wehi.edu.au./willson vectors.

The activity of these constructs was then tested as follows.

A. Fluorescence detection:

Single cell suspensions of M1 cells transfected with EFBOS SOCS1/GFP fusion constructs were washed in balanced salts solution (BSS) supplemented with 2% v/v fetal calf serum (FCS) and resuspended in 50 μl of BSS containing 2% v/v FCS and 1 μg/ml propidium iodide. Analyses were performed on a FACScan cell sorter (Bector-Dickinson) with dead cells excluded by propidium iodide (1 mg/ml) staining. The results showed that M1 cells expressing wild type GFP (M1 GFP 7.1.12), M1 cells expressing SOCS-1 as an N-terminal fusion with GFP (M1 SOCS-1-GFP 5.15) and a C-terminal fusion with GFP (M1 GFP-SOCS-1 6.46) were fluorescent, in contrast to a negative control clone.

B. Inhibition of M1 differentiation:

The capacity of M1 cells expressing SOCS1/GFP fusion proteins to differentiate in response to IL-6 was assessed in agar cultures. 200 cells in DMEM containing 20% v/v FCS and 0.3% w/v agar were plated in 1 ml cultures in 35 mm Petri dishes stimulated serially diluted concentrations of IL-6. Colony numbers and morphology were scored after 7 days incubation at 37° C. in a fully humidified atmosphere of 10% v/v CO₂ in air. Undifferentiated colonies were compact while colonies composed of dispersed cells or which had a halo of migrating cells around a central core, were scored as differentiated. Importantly, as well as being fluorescent, both SOCS-1/GFP fusion proteins were also able to inhibit IL-6 induced differentiation of M1 cells when stably expressed.

SOCS-1 has previously been shown to inhibit the in vitro kinase activity of JAK2. This experiment examined the ability of SOCS-1-GFP and GFP-SOCS-1 proteins to inhibit the in vitro kinase activity of JAK2. Flag-tagged JAK2 was, therefore, transiently expressed by electroporation in Cos cells with and without co-expression of SOCS-1 GFP and GFP-SOCS-1. After 48 hours were lysed on ice and JAK2 proteins immunoprecipitated using 5 ml anti-JAK2 antibody (UBI). Immunoprecipitates were washed, and subjected to an in vitro kinase assay as previously described. Proteins were then separated by SDS-PAGE on a 4-15% w/v gradient gel and the gel treated with KOH to remove phosphoserine and phosphothreonine. Incorporation of radiolabelled phosphate was detected using a phosphoimager. The SOCS-1-GFP and GFP-SOCS-1 chimeric proteins were able to inhibit JAC-2 kinase activity to the same extent at wild-type SOCS-1.

EXAMPLE 45 CONTROL OR SOCS GENE TRANSCRIPTION BY CYTOKINE

The inventors have shown that socs gene transcription is regulated by a range of cytokines. These studies have been extended by the Northern blot analysis of mRNA from organs of mice injected with a range of cytokine and cells treated with cytokines. SCOS-1 and SOCS-3 expression in ES cells is strictly controlled by LIF, whereas LIF does not appear to tightly regulate either expression of SOCS-2 or CIS. The expression of the SOCS-1, SOCS-2, SCOS-3 and CIS genes in bone marrow, spleen and lug occurs in response to a range of cytokines such as IL-2, IL-4, IL-5, IL-7, IL-9, IL-13, M-CSF, SCF, FL, EOP, TPO, anti-μ and LGH. Furthermore, the regulation of SCOS genes in vivo by clinically important cytokines is highlighted by the injection of GM-CSF into mice and analysis of the bonemarrow, spleen and lung at various times afterward. For SOCS-1, SOCS-2, SCOS-3 and CIS there is evidence of transcriptional control by GM-CSF.

EXAMPLE 46 CLONING OF SOCS cDNAs

DNA encoding the entire coding region of SCOS-13 has been constructed from cDNA and genomic DNA clones, Briefly, screening pulled out partial clones from both cDNA and genomic libraries, a full length coding region was generated by overlap PCR using a 5′ genomic fragment and a 3′ cDNa fragment, 5′ oligo to the genomic fragment was made with an Ascl site the 3′ oligo to the cDNa fragment was made with an MIul site so that the stitched together coding region could be ligated straight into pEF Bos flag construct. 5′ genomic SSB-1 Oligo No. 3342.

AGCT G GCG CGC C AG GGT CAG AAG GTC ACG GGA GGG [SEQ ID NO:58]      Asc 1        G     Q   K   V   T   G    G 3′ genomic oligo No 3243 AAG TCC GTT CAA GTA GCG CAT GCG GAT CTC [SEQ ID NO:52] 5′ cDNA Oligo No 3244 GAG ATC CGC ATG CGC TAC TTG AAC GGA CTT [SEQ ID NO:53]  E  I  R  M   R  Y  L   N   G   L (SEQ ID NO:54) 3′ cDNa Oligo No 3245 AGCT ACG CGT CTG GTA GAG GAG GTA GGC TTT GAG [SEQ ID NO:55]      Mlu1

The resulting nucleotide and predicted amino acid sequences are shown in FIGS. 49 and 50.

Similarly complete DNA and predicted amino acid sequences of SCOS-5 and SOCS-9 are shown in FIG. 51 A&B and 52 A&B.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

TABLE 4.1 Summary of ESTs derived from mouse SOCS-4 cDNAs SOCS Species EST name End EST no Library source Contig SOCS-4 Mouse mc65f04 5′ EST0549700 d13.5-14.5 mouse m4.1 mf42e06 5′ EST0593477 d13.5-14.5 mouse m4.1 embryo mp10c10 5′ EST0747905 d 8.5 mouse embryo m4.1 mr81g09 5′ HST0783081 d13 embryo m4.1 mt19h12 5′ EST0816531 spleen m4.1

TABLE 4.2 Summary of ESTs derived from human SOCS-4 cDNAS SOCS Species EST name End EST no Library source Contig SOCS-4 Human 27b5 5′ EST0534081 retina h4.2 30d2 5′ EST0534315 retina h4.2 J0159F 5′ EST0461188 foetal heart h4.2 J0159F 5′ EST0461428 foetal heart h4.2 EST19523 5′ EST0958884 retina h4.2 EST81149 5′ EST1011015 placenta h4.2 EST180909 5′ EST0951375 Jurkat T- h4.2 lymphocyte EST1S2619 5′ EST0953220 Jurkat T- h4.1 lymphocytc ya99h09 3′ EST0103262 placenta h4.2 ye70c04 5′ EST0172673 foeatal liver/spleen h4.2 yh53c09 5′ ES10197390 placenta h4.2 3′ EST0197391 h4.2 yh77g11 5′ EST0203418 placenta h4.2 3′ EST0203419 h4.1 yb87h05 5′ EST0204888 placenta h4.1 3′ EST0204773 h4.1 yi45h07 5′ EST0246604 placenta h4.2 yj04e06 5′ EST0258541 placenta h4.1 3′ EST0258285 h4.1 yq12h06 5′ EST0309968 foetal liver spleen h4.2 yq56a06 3′ EST0346924 foetal liver spleen h4.2 yq60e02 5′ EST0347259 foetal liver spleen h4.2 3′ EST0347209 h4.2 yq92g03 5′ EST0355932 foetal liver spleen h4.2 3′ EST0355884 h4.2 yq97h06 5′ EST0357618 foetal liver spleen h4.2 3′ EST0357416 h4.2 yr90f01 5′ EST0372402 foetal liver spleen h4.2 yt69c03 5′ EST0338395 foetal liver spleen h4.2 3′ EST0338303 h4.2 yv30a08 3′ EST0485806 foetal liver spleen h4.2 yv55f07 5′ EST0465391 foetal liver spleen h4.2 3′ EST0463331 h4.2 yv57h09 5′ EST0464336 foetal liver spleen h4.2 3′ EST0458765 h4.2 yv87h02 5′ EST0388085 melanocyte h4.2 yv98e11 5′ EST0400679 melanocyte h4.2 3′ EST0400680 h4.2 yw68d10 5′ EST0441370 placenta (8-9 wk) h4.2 yw82a03 5′ EST0463005 placenta (8-9 wk) h4.2 3′ EST0433678 h4.1 yx08a07 3′ EST0407016 melanoocyte h4.1 yx72h06 5′ EST0435158 melanoocyte h4.2 3′ EST0422871 melanoocyte h4.1 yx76b09 5′ EST0434011 melanoocyte h4.2 yy37h08 3′ EST0451704 melanoocyte h4.2 yy66b02 5′ EST0505446 multiple sclerosis h4.2 3′ lesion za81f08 5′ EST0511777 foetal lung h4.2 zb18f07 3′ EST0485315 foetal lung h4.1 zc06e08 5′ EST0540473 parathyroid tumor h4.1 3′ EST0540354 h4.1 zd14g06 3′ EST0564666 foetal heart h4.1 zd51h12 3′ EST0576099 foetal heart h4.1 zd52b09 5′ EST0562012 foetal heart h4.1 3′ EST0581958 h4.1 ze25g11 3′ EST0679543 foetal heart h4.1 ze69f02 5′ EST0635563 retina h4.2 3′ EST0635472 h4.1 zf54f03 5′ EST0680111 retina h4.2 zh96e07 5′ EST0616241 foetal liver spleen h4.2 3′ EST0615745 h4.2 zv66h12 5′ EST1043265 8-9 w foetus h4.2 zs83a08 5′ EST0920072 germinal centre B h4.I cell 3′ EST0920016 h4.1 zs83g08 5′ EST0920121 germinal centre B h4.1 cell 3′ EST0920122 h4.1

TABLE 5.1 Summary of ESTs derived from mouse SOCS-5 cDNAs SOCS Species EST name End EST no Library source Contig SOCS-5 Mouse mc55a01 5′ EST0541556 d13.5-14.5 mouse m5.1 embryo mh98f09 5′ EST0638237 placenta m5.1 my26h12 5′ EST0859939 mixed organs m5.1 ve24e06 5′ EST0819106 heart m5.1

TABLE 5.2 Summary of ESTs derived from human SOCS-5 cDNAs SOCS Species EST name End EST no Library source Contig SOCS-5 Human EST15B103 ? EST0255029 adipose tissue h5.1 EST15B105 ? EST0258028 adipose tissue h5.1 EST27530 5′ EST0965892 cerebellum h5.1 zf50f01 5′ EST0679820 retina h5.1

TABLE 6.1 Summary cf ESTs derived from mouse SOCS-6 cDNAS SOCS Species EST name End EST no Library source Contig SOCS-6 Mouse mc04c05 5′ EST0525832 d19.5 embryo m6.1 md4Sa03 5′ EST0566730 d13.5-14.5 embryo m6.1 mf31d03 5′ EST0675970 d13.5-14.5 embryo m6.1 mh26b07 5′ EST0628752 d13.5-14.5 placenta m6.1 mh78e11 5′ EST0637608 d13.5-14.5 placenta m6.1 mh88h09 5′ EST0644383 d13.5-14.5 placenta m6.1 mh94h07 5′ EST0638078 d13.5-14.5 placenta m6.1 mi27h04 5′ EST0644252 d13.5-14.5 embryo m6.I mj29c05 5′ EST0664093 d13.5-14.5 embryo m6.1 mp66g04 5′ EST0757905 thymus m6.1 mw75g03 5′ EST0847938 liver m6.1 va53b05 5′ EST0901540 d12.5 embryo m6.1 vb34h02 5′ EST0930132 lymph node m6.1 vc55d07 3′ EST1057735 2 cell embryo m6.1 vc59e05 3′ EST1058201 2 cell embryo m6.1 vc67d03 3′ EST1057849 2 cell embryo m6.1 vc68d10 3′ EST1058663 2 cell embryo m6.1 vc97h01 3′ EST1059343 2 cell embryo m6.1 vc99c08 3′ EST1059410 2 cell embryo m6.1 vd07h03 3′ EST1058173 2 cell embryo m6.1 vd08c01 3′ EST1058275 2 cell embryo m6.1 vd09b12 3′ EST1058632 2 cell embryo m6.1 vd19b02 3′ EST1059723 2 cell embryo m6.1 vd29a04 3′ ? none found m6.1 vd46d06 3′ ? none found m6.1

TABLE 6.2 Summary of ESTs derived from human SOCS-5 cDNAs SOCS Species EST name End EST no Library source Contig SOCS-6 Human yf61e08 5′ EST0184387 d73 infant brain h6.1 yf93a09 5′ EST0186084 d73 infant brain h6.1 yg05f12 5′ EST0191486 d73 infant brain h6.1 yg41f04 5′ EST0195017 d73 infant brain h6.1 yg45c02 5′ EST0185308 d73 infant brain h6.1 yh11f10 5′ EST0236705 d73 infant brain h6.1 yh13b05 5′ EST0237191 d73 infant brain h6.1 3′ EST0236958 h6.2 ze35a12 5′ EST0555518 senescent h6.1 fibroblasts ze02h08 5′ EST0603826 foetal heart h6.1 3′ EST0603716 h6.2 z109a03 5′ EST0773936 pregnant uterus h6.1 3′ EST0773892 h6.1 z169e10 5′ EST0683363 colon h6.1 zn39d08 5′ EST0718885 endothelial cell h6.1 zo39e06 5′ EST0785947 endothelial cell h6.1

TABLE 7.1 Summary of ESTs derived from mouse SOCS-7 cDNAs SOCS Species EST name End EST no Library source Contig SOCS-7 Mouse mj39a01 5′ EST0665627 d13.5/14/5 embryo m7.1 vi52h07 5′ EST1267404 d7.5 embryo m7.1

TABLE 7-2 Summary of ESTs derived from human SOCS-5 cDNAs SOCS Species EST name End EST no Library source Contig SOCS-7 HUMAN STS WI-30171 (G21563) Chromosome 2 h7.2 EST00939 5′ EST0000906 hippocampus b7.1 EST12913 3′ EST0944382 uterus h7.2 yc29b05 3′ EST0128727 liver h7.2 yp49f10 3′ EST0301914 retina h7.2 xt10f03 5′ EST0922932 germinal centre h7.2 B cell 3′ EST0921231 h7.1 zx73g04 3′ EST1102975 ovarian tumour h7.1

TABLE 8.1 Summary of ESTs derived from mouse SOCS-8 cDNAs SOCS Species EST name End EST no Library source Contig SOCS-8 Mouse mj16e09 rl EST0666240 d13.5/14.5 embryo m8.1 vj27a029 rl EST1155973 heart m8.1

TABLE 9.1 Summary of ESTs derived from mouse SOCS-9 cDNAs SOCS Species EST name End EST no Library source Contig Mouse me65d05 5′ EST0585211 d13.5/14.5 embryo m9.1

TABLE 9.2 Summary of ESTs derived from human SOCS-5 cDNAs SOCS Species EST name End EST no Library source Contig SOCS-9 Human CSRL-83f2-u (B06659) chromosome 11 h9.1 EST114054 5′ EST0939759 placenta h9.1 yy06b07 3′ EST0434504 melanocyte h9.1 yy06g06 5′ EST0443783 melanocyte h9.1 zr40c09 5′ EST0832461 melanocyte, heart, h9.1 uterus zr72h01 5′ EST0892025 melanocyte, heart, h9.1 uterus 3′ EST0892026 h9.1 yx92c08 5′ EST0441160 melanocyte h9.1 yx93b08 5′ EST0441260 melanocyte h9.1 hfe0662 5′ EST0889611 foetal heart h9.1

TABLE 10.1 Summary of ESTs derived from mouse SOCS-10 cDNAs SOCS Species EST name End EST no Library source Contig Mouse mb14d12 5′ EST0549887 d19.5 embryo m10.1 mb40f06 5′ EST0515064 d19.5 embryo m19.1 mg89b11 5′ EST0630631 d13.5-145 embryo m10.1 mq89e12 5′ EST0776015 heart m10.1 mp03g12 5′ EST0741991 heart m10.1 vh53c11 5′ EST1154634 mammary gland m10.1

TABLE 10.2 Summary of ESTs derived from human SOCS-5 cDNAs SOCS Species EST name End EST no Library source Contig SOCS.10 Human aa48h10 3′ EST1135220 germinal centre h10.2 B cell zp35h01 3′ EST0819137 muscle h10.2 zp97h12 5′ EST0835442 muscle h10.2 3′ EST0531211 h10.2 zq08h01 5′ EST0835907 muscle h10.1 zr34g05 5′ EST0834251 melanocyte, heart, h10.2 uterus 3′ EST0834440 h10.2 EST73000 5 EST1004491 ovary h10.2 HSDHE1005 ? EST0013906 heart h10.2

TABLE 11.1 Summary of ESTs derived from human SOCS-5 cDNAs SOCS Species EST name End EST no Library source Contig SOCS-11 Human zt24h06 rl EST0925023 ovarian tumor 11.1 zr43b02 rl EST0873006 melanocyte, heart, 11.1 uterus sl EST0872954 11.1

TABLE 12.1 Summary of ESTs derived from mouse SOCS-12 cDNAs SOCS Species EST name End EST no Library source Contig SOCS-12 Mouse EST03803 5′ EST1054173 day 7.5 emb m12.1 ectoplacental cone mt18f02 5′ EST0817552 3NbMS spleen m12.1 mz60g10 5′ EST0690672 lymph node m12.1 va05c11 5′ EST0909449 lymph node m12.1

TABLE 12.2 Summary of ESTs derived from human SOCS-5 cDNAs SOCS Species EST name End EST no Library source Contig SOCS-12 Human STS-SHGC-13867 Chromosome 2 h12.2 EST177695 5′ EST0948071 Jurkat cells h12.1 EST64550 5′ EST0997367 Jurkat cells h12.1 EST76868 5′ EST1007291 pineal body h12.2 PMY2369 5′ EST1115998 KG-1 h12.1 yb38f04 5′ EST0108807 foetal spleen h12.1 3′ h12.2 yg74c12 5′ EST0224407 d73 brain h12.1 yh13g04 5′ EST0237226 d73 brain h12.1 3′ EST0236992 h12.2 yh48b06 5′ yh48b06 placenta h12.2 yh53a05 5′ EST0197282 placenta h12.2 3′ EST0197486 h12.2 yn48h09 5′ EST0278259 brain h12.2 3′ EST0278259 h12.2 yn90a09 3′ EST0302557 brain h12.2 yo08f03 5′ EST0301790 brain h12.2 3′ EST0302059 h12.2 yo11e01 3′ ?none found h12.2 yo63b12 5′ EST0303606 breast h12.2 3′ EST0304085 h12.2 yq56g02 3′ EST0346935 foetal liver spleen h12.1 zh57c04 3′ EST0594201 foetal liver spleen h12.2 7h79h01 3′ EST0598945 foetal liver spleen h12.2 zh99a11 3′ EST0618570 foetal liver spleen h12.2 zo92h12 5′ EST0803392 ovarian cancer h12.1 3′ EST0803393 h12.2 zs48c01 5′ EST0925714 germinal centre h12.1 B cell 3′ EST0925530 h12.2 zs45h02 3′ EST0932296 germinal centre h12.2 B cell

TABLE 13.1 Summary of ESTs derived from mouse SOCS-13 cDNAs SOCS Species EST name End EST no Library source Contig SOCS-13 Mouse ma39c09 5′ EST0517875 day 19.5 embryo m13.1 mc60c05 5′ EST0584950 day 13.5/14.5 embryo m13.1 mi78g05 5′ EST0653834 day 19.5 embryo m13.1 mk10c11 5′ EST0735158 day 19.5 embryo m13.1 mo48g12 5′ EST0745111 day 10.5 embryo m13.1 mp94a01 5′ EST0762827 thymus m13.1 vb57c07 5′ EST1028976 day 11.5 embryo m13.1 vh07c11 5 EST1117269 mammary gland m13.1

TABLE 13.2 Summary of ESTs derived from human SOCS-13 cDNAS SOCS Species EST name End EST no Library source Contig SOCS-13 Human EST59161 5′ EST0992726 infant brain h13.1

TABLE 14.1 Summary of ESTs derived from mouse SOCS-14 cDNAs SOCS Species EST name End EST no Library source Contig SOCS-14 mouse mi75e03 5′ EST0651892 d19.5 embryo m14.1 vd29h11 5′ EST1067080 2 cell embryo m14.1 vd53g07 5′ EST1119627 2 cell embryo m14.1

TABLE 15.1 Summary of ESTs derived from mouse SOCS-15 cDNAs SOCS Species EST name End EST no Library source Contig SOCS-15 Mouse mh29b05 5′ EST062S834 placenta m15.1 mh98h09 5′ EST0638243 placenta m15.l m145a02 5′ EST0687171 testis m15.1 mu43a10 5′ EST851588 thymus m15.l my38c09 5′ EST878461 pooled organs m15.1 vj37h07 5′ EST1174791 diaphragm m15.1 AC002393 5′ Chromosome 6 m15.1 BAC

TABLE 15.2 Summary of ESTs derived from human SOCS-15 cDNAs SOCS Species EST name End EST no Library source Contig SOCS-15 Human EST98889 5′ EST1026568 thyroid h15.1 ne48bo5 3′ EST1138057 colon tumour h15.1 yb12h12 5′ EST0098885 placenta h15.1 3′ EST0098886 h15.1 HSU47924 Chromosome 12 h15.1 BAC

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SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 81 <210> SEQ ID NO 1 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic <400> SEQUENCE: 1 cacgccgccc acgtgaaggc 20 <210> SEQ ID NO 2 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic <400> SEQUENCE: 2 ttcgccaatg acaagacgct 20 <210> SEQ ID NO 3 <211> LENGTH: 1235 <212> TYPE: DNA <213> ORGANISM: Mouse <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (161)..(796) <400> SEQUENCE: 3 cgaggctcaa gctccgggcg gattctgcgt gccgctctcg ctccttgggg tctgttggcc 60 ggcctgtgcc acccggacgc ccggctcact gcctctgtct cccccatcag cgcagccccg 120 gacgctatgg cccacccctc cagctggccc ctcgagtagg atg gta gca cgc aac 175 Met Val Ala Arg Asn 1 5 cag gtg gca gcc gac aat gcg atc tcc ccg gca gca gag ccc cga cgg 223 Gln Val Ala Ala Asp Asn Ala Ile Ser Pro Ala Ala Glu Pro Arg Arg 10 15 20 cgg tca gag ccc tcc tcg tcc tcg tct tcg tcc tcg cca gcg gcc ccc 271 Arg Ser Glu Pro Ser Ser Ser Ser Ser Ser Ser Ser Pro Ala Ala Pro 25 30 35 gtg cgt ccc cgg ccc tgc ccg gcg gtc cca gcc cca gcc cct ggc gac 319 Val Arg Pro Arg Pro Cys Pro Ala Val Pro Ala Pro Ala Pro Gly Asp 40 45 50 act cac ttc cgc acc ttc cgc tcc cac tcc gat tac cgg cgc atc acg 367 Thr His Phe Arg Thr Phe Arg Ser His Ser Asp Tyr Arg Arg Ile Thr 55 60 65 cgg acc agc gcg ctc ctg gac gcc tgc ggc ttc tat tgg gga ccc ctg 415 Arg Thr Ser Ala Leu Leu Asp Ala Cys Gly Phe Tyr Trp Gly Pro Leu 70 75 80 85 agc gtg cac ggg gcg cac gag cgg ctg cgt gcc gag ccc gtg ggc acc 463 Ser Val His Gly Ala His Glu Arg Leu Arg Ala Glu Pro Val Gly Thr 90 95 100 ttc ttg gtg cgc gac agt cgt caa cgg aac tgc ttc ttc gcg ctc agc 511 Phe Leu Val Arg Asp Ser Arg Gln Arg Asn Cys Phe Phe Ala Leu Ser 105 110 115 gtg aag atg gct tcg ggc ccc acg agc atc cgc gtg cac ttc cag gcc 559 Val Lys Met Ala Ser Gly Pro Thr Ser Ile Arg Val His Phe Gln Ala 120 125 130 ggc cgc ttc cac ttg gac ggc agc cgc gag acc ttc gac tgc ctt ttc 607 Gly Arg Phe His Leu Asp Gly Ser Arg Glu Thr Phe Asp Cys Leu Phe 135 140 145 gag ctg ctg gag cac tac gtg gcg gcg ccg cgc cgc atg ttg ggg gcc 655 Glu Leu Leu Glu His Tyr Val Ala Ala Pro Arg Arg Met Leu Gly Ala 150 155 160 165 ccg ctg cgc cag cgc cgc gtg cgg ccg ctg cag gag ctg tgt cgc cag 703 Pro Leu Arg Gln Arg Arg Val Arg Pro Leu Gln Glu Leu Cys Arg Gln 170 175 180 cgc atc gtg gcc gcc gtg ggt cgc gag aac ctg gcg cgc atc cct ctt 751 Arg Ile Val Ala Ala Val Gly Arg Glu Asn Leu Ala Arg Ile Pro Leu 185 190 195 aac ccg gta ctc cgt gac tac ctg agt tcc ttc ccc ttc cag atc 796 Asn Pro Val Leu Arg Asp Tyr Leu Ser Ser Phe Pro Phe Gln Ile 200 205 210 tgaccggctg ccgctgtgcc gcagcattaa gtgggggcgc cttattattt cttattatta 856 attattatta tttttctgga accacgtggg agccctcccc gcctgggtcg gagggagtgg 916 ttgtggaggg tgagatgcct cccacttctg gctggagacc tcatcccacc tctcaggggt 976 gggggtgctc ccctcctggt gctccctccg ggtcccccct ggttgtagca gcttgtgtct 1036 ggggccagga cctgaattcc actcctacct ctccatgttt acatattccc agtatctttg 1096 cacaaaccag gggtcgggga gggtctctgg cttcattttt ctgctgtgca gaatatccta 1156 ttttatattt ttacagccag tttaggtaat aaactttatt atgaaagttt ttttttaaaa 1216 gaaaaaaaaa aaaaaaaaa 1235 <210> SEQ ID NO 4 <211> LENGTH: 212 <212> TYPE: PRT <213> ORGANISM: Mouse <400> SEQUENCE: 4 Met Val Ala Arg Asn Gln Val Ala Ala Asp Asn Ala Ile Ser Pro Ala 1 5 10 15 Ala Glu Pro Arg Arg Arg Ser Glu Pro Ser Ser Ser Ser Ser Ser Ser 20 25 30 Ser Pro Ala Ala Pro Val Arg Pro Arg Pro Cys Pro Ala Val Pro Ala 35 40 45 Pro Ala Pro Gly Asp Thr His Phe Arg Thr Phe Arg Ser His Ser Asp 50 55 60 Tyr Arg Arg Ile Thr Arg Thr Ser Ala Leu Leu Asp Ala Cys Gly Phe 65 70 75 80 Tyr Trp Gly Pro Leu Ser Val His Gly Ala His Glu Arg Leu Arg Ala 85 90 95 Glu Pro Val Gly Thr Phe Leu Val Arg Asp Ser Arg Gln Arg Asn Cys 100 105 110 Phe Phe Ala Leu Ser Val Lys Met Ala Ser Gly Pro Thr Ser Ile Arg 115 120 125 Val His Phe Gln Ala Gly Arg Phe His Leu Asp Gly Ser Arg Glu Thr 130 135 140 Phe Asp Cys Leu Phe Glu Leu Leu Glu His Tyr Val Ala Ala Pro Arg 145 150 155 160 Arg Met Leu Gly Ala Pro Leu Arg Gln Arg Arg Val Arg Pro Leu Gln 165 170 175 Glu Leu Cys Arg Gln Arg Ile Val Ala Ala Val Gly Arg Glu Asn Leu 180 185 190 Ala Arg Ile Pro Leu Asn Pro Val Leu Arg Asp Tyr Leu Ser Ser Phe 195 200 205 Pro Phe Gln Ile 210 <210> SEQ ID NO 5 <211> LENGTH: 1121 <212> TYPE: DNA <213> ORGANISM: Mouse <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (223)..(816) <400> SEQUENCE: 5 gcgatctgtg ggtgacagtg tctgcgagag actttgccac accattctgc cggaatttgg 60 agaaaaagaa ccagccgctt ccagtcccct ccccctccgc caccatttcg gacaccctgc 120 acactctcgt tttggggtac cctgtgactt ccaggcagca cgcgaggtcc actggcccca 180 gctcgggcga ccagctgtct gggacgtgtt gactcatctc cc atg acc ctg cgg 234 Met Thr Leu Arg 1 tgc ctg gag ccc tcc ggg aat gga gcg gac agg acg cgg agc cag tgg 282 Cys Leu Glu Pro Ser Gly Asn Gly Ala Asp Arg Thr Arg Ser Gln Trp 5 10 15 20 ggg acc gcg ggg ttg ccg gag gaa cag tcc ccc gag gcg gcg cgt ctg 330 Gly Thr Ala Gly Leu Pro Glu Glu Gln Ser Pro Glu Ala Ala Arg Leu 25 30 35 gcg aaa gcc ctg cgc gag ctc agt caa aca gga tgg tac tgg gga agt 378 Ala Lys Ala Leu Arg Glu Leu Ser Gln Thr Gly Trp Tyr Trp Gly Ser 40 45 50 atg act gtt aat gaa gcc aaa gag aaa tta aaa gag gct cca gaa gga 426 Met Thr Val Asn Glu Ala Lys Glu Lys Leu Lys Glu Ala Pro Glu Gly 55 60 65 act ttc ttg att aga gat agt tcg cat tca gac tac cta cta act ata 474 Thr Phe Leu Ile Arg Asp Ser Ser His Ser Asp Tyr Leu Leu Thr Ile 70 75 80 tcc gtt aag acg tca gct gga ccg act aac ctg cgg att gag tac caa 522 Ser Val Lys Thr Ser Ala Gly Pro Thr Asn Leu Arg Ile Glu Tyr Gln 85 90 95 100 gat ggg aaa ttc aga ttg gat tct atc ata tgt gtc aag tcc aag ctt 570 Asp Gly Lys Phe Arg Leu Asp Ser Ile Ile Cys Val Lys Ser Lys Leu 105 110 115 aaa cag ttt gac agt gtg gtt cat ctg att gac tac tat gtc cag atg 618 Lys Gln Phe Asp Ser Val Val His Leu Ile Asp Tyr Tyr Val Gln Met 120 125 130 tgc aag gat aaa cgg aca ggc cca gaa gcc cca cgg aat ggg act gtt 666 Cys Lys Asp Lys Arg Thr Gly Pro Glu Ala Pro Arg Asn Gly Thr Val 135 140 145 cac ctg tac ctg acc aaa cct ctg tat aca tca gca ccc act ctg cag 714 His Leu Tyr Leu Thr Lys Pro Leu Tyr Thr Ser Ala Pro Thr Leu Gln 150 155 160 cat ttc tgt cga ctc gcc att aac aaa tgt acc ggt acg atc tgg gga 762 His Phe Cys Arg Leu Ala Ile Asn Lys Cys Thr Gly Thr Ile Trp Gly 165 170 175 180 ctg cct tta cca aca aga cta aaa gat tac ttg gaa gaa tat aaa ttc 810 Leu Pro Leu Pro Thr Arg Leu Lys Asp Tyr Leu Glu Glu Tyr Lys Phe 185 190 195 cag gta taagtatttc tctctctttt tcgttttttt ttaaaaaaaa aaaaacacat 866 Gln Val gcctcatata gactatctcc gaatgcagct atgtgaaaga gaacccagag gccctcctct 926 ggataactgc gcagaattct ctcttaagga cagttgggct cagtctaact taaaggtgtg 986 aagatgtagc taggtatttt aaagttcccc ttaggtagtt ttagctgaat gatgctttct 1046 ttcctatggc tgctcaagat caaatggccc ttttaaatga aacaaaacaa aacaaaacaa 1106 aaaaaaaaaa aaaaa 1121 <210> SEQ ID NO 6 <211> LENGTH: 198 <212> TYPE: PRT <213> ORGANISM: Mouse <400> SEQUENCE: 6 Met Thr Leu Arg Cys Leu Glu Pro Ser Gly Asn Gly Ala Asp Arg Thr 1 5 10 15 Arg Ser Gln Trp Gly Thr Ala Gly Leu Pro Glu Glu Gln Ser Pro Glu 20 25 30 Ala Ala Arg Leu Ala Lys Ala Leu Arg Glu Leu Ser Gln Thr Gly Trp 35 40 45 Tyr Trp Gly Ser Met Thr Val Asn Glu Ala Lys Glu Lys Leu Lys Glu 50 55 60 Ala Pro Glu Gly Thr Phe Leu Ile Arg Asp Ser Ser His Ser Asp Tyr 65 70 75 80 Leu Leu Thr Ile Ser Val Lys Thr Ser Ala Gly Pro Thr Asn Leu Arg 85 90 95 Ile Glu Tyr Gln Asp Gly Lys Phe Arg Leu Asp Ser Ile Ile Cys Val 100 105 110 Lys Ser Lys Leu Lys Gln Phe Asp Ser Val Val His Leu Ile Asp Tyr 115 120 125 Tyr Val Gln Met Cys Lys Asp Lys Arg Thr Gly Pro Glu Ala Pro Arg 130 135 140 Asn Gly Thr Val His Leu Tyr Leu Thr Lys Pro Leu Tyr Thr Ser Ala 145 150 155 160 Pro Thr Leu Gln His Phe Cys Arg Leu Ala Ile Asn Lys Cys Thr Gly 165 170 175 Thr Ile Trp Gly Leu Pro Leu Pro Thr Arg Leu Lys Asp Tyr Leu Glu 180 185 190 Glu Tyr Lys Phe Gln Val 195 <210> SEQ ID NO 7 <211> LENGTH: 2187 <212> TYPE: DNA <213> ORGANISM: Mouse <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (18)..(692) <400> SEQUENCE: 7 cgctggctcc gtgcgcc atg gtc acc cac agc aag ttt ccc gcc gcc ggg 50 Met Val Thr His Ser Lys Phe Pro Ala Ala Gly 1 5 10 atg agc cgc ccc ctg gac acc agc ctg cgc ctc aag acc ttc agc tcc 98 Met Ser Arg Pro Leu Asp Thr Ser Leu Arg Leu Lys Thr Phe Ser Ser 15 20 25 aaa agc gag tac cag ctg gtg gtg aac gcc gtg cgc aag ctg cag gag 146 Lys Ser Glu Tyr Gln Leu Val Val Asn Ala Val Arg Lys Leu Gln Glu 30 35 40 agc gga ttc tac tgg agc gcc gtg acc ggc ggc gag gcg aac ctg ctg 194 Ser Gly Phe Tyr Trp Ser Ala Val Thr Gly Gly Glu Ala Asn Leu Leu 45 50 55 ctc agc gcc gag ccc gcg ggc acc ttt ctt atc cgc gac agc tcg gac 242 Leu Ser Ala Glu Pro Ala Gly Thr Phe Leu Ile Arg Asp Ser Ser Asp 60 65 70 75 cag cgc cac ttc ttc acg ttg agc gtc aag acc cag tcg ggg acc aag 290 Gln Arg His Phe Phe Thr Leu Ser Val Lys Thr Gln Ser Gly Thr Lys 80 85 90 aac cta cgc atc cag tgt gag ggg ggc agc ttt tcg ctg cag agt gac 338 Asn Leu Arg Ile Gln Cys Glu Gly Gly Ser Phe Ser Leu Gln Ser Asp 95 100 105 ccc cga agc acg cag cca gtt ccc cgc ttc gac tgt gta ctc aag ctg 386 Pro Arg Ser Thr Gln Pro Val Pro Arg Phe Asp Cys Val Leu Lys Leu 110 115 120 gtg cac cac tac atg ccg cct cca ggg acc ccc tcc ttt tct ttg cca 434 Val His His Tyr Met Pro Pro Pro Gly Thr Pro Ser Phe Ser Leu Pro 125 130 135 ccc acg gaa ccc tcg tcc gaa gtt ccg gag cag cca cct gcc cag gca 482 Pro Thr Glu Pro Ser Ser Glu Val Pro Glu Gln Pro Pro Ala Gln Ala 140 145 150 155 ctc ccc ggg agt acc ccc aag aga gct tac tac atc tat tct ggg ggc 530 Leu Pro Gly Ser Thr Pro Lys Arg Ala Tyr Tyr Ile Tyr Ser Gly Gly gag aag att ccg ctg gta ctg agc cga cct ctc tcc tcc aac gtg gcc 578 Glu Lys Ile Pro Leu Val Leu Ser Arg Pro Leu Ser Ser Asn Val Ala 175 180 185 acc ctc cag cat ctt tgt cgg aag act gtc aac ggc cac ctg gac tcc 626 Thr Leu Gln His Leu Cys Arg Lys Thr Val Asn Gly His Leu Asp Ser 190 195 200 tat gag aaa gtg acc cag ctg cct gga ccc att cgg gag ttc ctg gat 674 Tyr Glu Lys Val Thr Gln Leu Pro Gly Pro Ile Arg Glu Phe Leu Asp 205 210 215 cag tat gat gct cca ctt taaggagcaa aagggtcaga ggggggcctg 722 Gln Tyr Asp Ala Pro Leu 220 225 ggtcggtcgg tcgcctctcc tccgaggcac atggcacaag cacaaaaatc cagccccaac 782 ggtcggtagc tcccagtgag ccaggggcag attggcttct tcctcaggcc ctccactccc 842 gcagagtaga gctggcagga cctggaattc gtctgagggg agggggagct gccacctgct 902 ttcccccctc ccccagctcc agcttctttc aagtggagcc agccggcctg gcctggtggg 962 acaatacctt tgacaagcgg actctcccct ccccttcctc cacaccccct ctgcttccca 1022 agggaggtgg ggacacctcc aagtgttgaa cttagaactg caaggggaat cttcaaactt 1082 tcccgctgga acttgtttgc gctttgattt ggtttgatca agagcaggca cctgggggaa 1142 ggatggaaga gaaaagggtg tgtgaagggt ttttatgctg gccaaagaaa taaccactcc 1202 cactgcccaa cctaggtgag gagtggtggc tcctggctct ggggagagtg gcaaggggtg 1262 acctgaagag agctatactg gtgccaggct cctctccatg gggcagctaa tgaaacctcg 1322 cagatccctt gcaccccaga accctccccg ttgtgaagag gcagtagcat ttagaaggga 1382 gacagatgag gctggtgagc tggccgcctt ttccaacacc gaagggaggc agatcaacag 1442 atgagccatc ttggagccca ggtttcccct ggagcagatg gagggttctg ctttgtctct 1502 cctatgtggg gctaggagac tcgccttaaa tgccctctgt cccagggatg gggattggca 1562 cacaaggagc caaacacagc caataggcag agagttgagg gattcaccca ggtggctaca 1622 ggccagggga agtggctgca ggggagagac ccagtcactc caggagactc ctgagttaac 1682 actgggaaga cattggccag tcctagtcat ctctcggtca gtaggtccga gagcttccag 1742 gccctgcaca gccctccttt ctcacctggg gggaggcagg aggtgatgga gaagccttcc 1802 catgccgctc acaggggcct cacgggaatg cagcagccat gcaattacct ggaactggtc 1862 ctgtgttggg gagaaacaag ttttctgaag tcaggtatgg ggctgggtgg ggcagctgtg 1922 tgttggggtg gcttttttct ctctgttttg aataatgttt acaatttgcc tcaatcactt 1982 ttataaaaat ccacctccag cccgcccctc tccccactca ggccttcgag gctgtctgaa 2042 gatgcttgaa aaactcaacc aaatcccagt tcaactcaga ctttgcacat atatttatat 2102 ttatactcag aaaagaaaca tttcagtaat ttataataaa agagcactat tttttaatga 2162 aaaaaaaaaa aaaaaaaaaa aaaaa 2187 <210> SEQ ID NO 8 <211> LENGTH: 225 <212> TYPE: PRT <213> ORGANISM: Mouse <400> SEQUENCE: 8 Met Val Thr His Ser Lys Phe Pro Ala Ala Gly Met Ser Arg Pro Leu 1 5 10 15 Asp Thr Ser Leu Arg Leu Lys Thr Phe Ser Ser Lys Ser Glu Tyr Gln 20 25 30 Leu Val Val Asn Ala Val Arg Lys Leu Gln Glu Ser Gly Phe Tyr Trp 35 40 45 Ser Ala Val Thr Gly Gly Glu Ala Asn Leu Leu Leu Ser Ala Glu Pro 50 55 60 Ala Gly Thr Phe Leu Ile Arg Asp Ser Ser Asp Gln Arg His Phe Phe 65 70 75 80 Thr Leu Ser Val Lys Thr Gln Ser Gly Thr Lys Asn Leu Arg Ile Gln 85 90 95 Cys Glu Gly Gly Ser Phe Ser Leu Gln Ser Asp Pro Arg Ser Thr Gln 100 105 110 Pro Val Pro Arg Phe Asp Cys Val Leu Lys Leu Val His His Tyr Met 115 120 125 Pro Pro Pro Gly Thr Pro Ser Phe Ser Leu Pro Pro Thr Glu Pro Ser 130 135 140 Ser Glu Val Pro Glu Gln Pro Pro Ala Gln Ala Leu Pro Gly Ser Thr 145 150 155 160 Pro Lys Arg Ala Tyr Tyr Ile Tyr Ser Gly Gly Glu Lys Ile Pro Leu 165 170 175 Val Leu Ser Arg Pro Leu Ser Ser Asn Val Ala Thr Leu Gln His Leu 180 185 190 Cys Arg Lys Thr Val Asn Gly His Leu Asp Ser Tyr Glu Lys Val Thr 195 200 205 Gln Leu Pro Gly Pro Ile Arg Glu Phe Leu Asp Gln Tyr Asp Ala Pro 210 215 220 Leu 225 <210> SEQ ID NO 9 <211> LENGTH: 1094 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 9 ctccggctgg ccccttctgt aggatggtag cacacaacca ggtggcagcc gacaatgcag 60 tctccacagc agcagagccc cgacggcggc cagaaccttc ctcctcttcc tcctcctcgc 120 ccgcggcccc cgcgcgcccg cggccgtgcc ccgcggtccc ggccccggcc cccggcgaca 180 cgcacttccg cacattccgt tcgcacgccg attaccggcg catcacgcgc gccagcgcgc 240 tcctggacgc ctgcggattc tactgggggc ccctgagcgt gcacggggcg cacgagcggc 300 tgcgcgccga gcccgtgggc accttcctgg tgcgcgacag ccgccagcgg aactgctttt 360 tcgcccttag cgtgaagatg gcctcgggac ccacgagcat ccgcgtgcac tttcaggccg 420 gccgctttca cctggatggc agccgcgaga gcttcgactg cctcttcgag ctgctggagc 480 actacgtggc ggcgccgcgc cgcatgctgg gggccccgct gcgccagcgc cgcgtgcggc 540 cgctgcagga gctgtgccgc cagcgcatcg tggccaccgt gggccgcgag aacctggctc 600 gcatccccct caaccccgtc ctccgcgact acctgagctc cttccccttc cagatttgac 660 cggcagcgcc cgccgtgcac gcagcattaa ctgggatgcc gtgttatttt gttattactt 720 gcctggaacc atgtgggtac cctccccggc ctgggttgga gggagcggat gggtgtaggg 780 gcgaggcgcc tcccgccctc ggctggagac gaggccgcag accccttctc acctcttgag 840 ggggtcctcc ccctcctggt gctccctctg ggtccccctg gttgttgtag cagcttaact 900 gtatctggag ccaggacctg aactcgcacc tcctacctct tcatgtttac atatacccag 960 tatctttgca caaaccaggg gttgggggag ggtctctggc tttatttttc tgctgtgcag 1020 aatcctattt tatatttttt aaagtcagtt taggtaataa actttattat gaaagttttt 1080 ttttttaaaa aaaa 1094 <210> SEQ ID NO 10 <211> LENGTH: 211 <212> TYPE: PRT <213> ORGANISM: Human <400> SEQUENCE: 10 Met Val Ala His Asn Gln Val Ala Ala Asp Asn Ala Val Ser Thr Ala 1 5 10 15 Ala Glu Pro Arg Arg Arg Pro Glu Pro Ser Ser Ser Ser Ser Ser Ser 20 25 30 Pro Ala Ala Pro Ala Arg Pro Arg Pro Cys Pro Ala Val Pro Ala Pro 35 40 45 Ala Pro Gly Asp Thr His Phe Arg Thr Phe Arg Ser His Ala Asp Tyr 50 55 60 Arg Arg Ile Thr Arg Ala Ser Ala Leu Leu Asp Ala Cys Gly Phe Tyr 65 70 75 80 Trp Gly Pro Leu Ser Val His Gly Ala His Glu Arg Leu Arg Ala Glu 85 90 95 Pro Val Gly Thr Phe Leu Val Arg Asp Ser Arg Gln Arg Asn Cys Phe 100 105 110 Phe Ala Leu Ser Val Lys Met Ala Ser Gly Pro Thr Ser Ile Arg Val 115 120 125 His Phe Gln Ala Gly Arg Phe His Leu Asp Gly Ser Arg Glu Ser Phe 130 135 140 Asp Cys Leu Phe Glu Leu Leu Glu His Tyr Val Ala Ala Pro Arg Arg 145 150 155 160 Met Leu Gly Ala Pro Leu Arg Gln Arg Arg Val Arg Pro Leu Gln Glu 165 170 175 Leu Cys Arg Gln Arg Ile Val Ala Thr Val Gly Arg Glu Asn Leu Ala 180 185 190 Arg Ile Pro Leu Asn Pro Val Leu Arg Asp Tyr Leu Ser Ser Phe Pro 195 200 205 Phe Gln Ile 210 <210> SEQ ID NO 11 <211> LENGTH: 2807 <212> TYPE: DNA <213> ORGANISM: Rat <400> SEQUENCE: 11 ggaaaccgag gcggggagac caggaggcct tggcctcaga gcttcagagt cgcgtggcag 60 caaacagaga aacctgtaga gggcagtgtg cgtcacttag ctcagggaag ctgcacgcga 120 aactcacccg ccttcattca taaacatcgt cagctaggca cctactcctg ggctttcagg 180 acaaactgaa tcacgaaacc acagtgtcct taaaataggt ctgaccgcct gaatccctgg 240 ccaaggtgtg tacggggcat gggagccctt gtgcagagat gcttgcagga gccttgaggg 300 gctctgtaag acagaggcta ggaagacaaa gttgggggct acagcttctt gtcctgcccg 360 gggcctcagt ttcttcggtt gcccacgtag gagtgcagag agtccagccc ctggggaccc 420 aacccaaccc cgcccagttt ccgaggaact cgtccgggag cgggggcgcc cctcccgcac 480 cgccttaggc ttcctttgaa gcctctgcgg tcaggccacc gcttcctggg aagcccaagc 540 caaggccagg ccgagtggcc aacgggaggg gcccgcgcgc gattctggag gagggcggcg 600 gccccacagg tctccagggc tggctagccg ggctcctaga gcggagactg ccaaggcctt 660 cgggtcctgg gcaggaagga tcctggcagg gaggagttgc ttggggggtg ggggggaaag 720 gctccaggcg cggtggagct ctgaccagga gaatgcacac actcggaggg gaggaggcgt 780 gtcagcccca agctagcatc ccacccgggg agcagcgatg tggggcgaag gtagccagag 840 caaaagagca ggcaccaggt gacacgaaac agaagattcc gggtagagcc agaaccccag 900 aagtcccatt cagggaaggt gcgaggcgag aacgagttag gtggaccctc tccaggggca 960 gccaaagaaa tctaaagaga acccgaagga cttgccggaa agagaaaccg aaagcggcgg 1020 tgggcgggat cggtgggcgg ggcctccctg gtttaagagc ttgatgcagg ggcgggcagc 1080 agcagagaga actgcggccg tggcagcggc acggctcccg gccccggagc atgcgcgaca 1140 gcagccccgg aacccccagc cgcggcgccc cgcgtcccgc cgccaggtga gccgaggcag 1200 ctgcgaagga gcaggcggga ggggatggga ggaaggggag cagagcctgg caggactatc 1260 ctcgcagact gcatggcggg gtcgtggatg ctatgcctct ggcgcccgcc ccaccggctg 1320 gcccaggcgg cccctcgcgc gcgcggggcg ccgtcagccc ctcctctccg gccctgagcc 1380 cggatcgtcc gcccgggttc cagttcccgg cgtggccagt aggcggcaac cgcgaggcgg 1440 caagccaccc agcggggacg gcctggagtc gggcccctct ccacgccccc ttctccacgc 1500 gcgcggggag gcagggctcc accgccagtc tggaagggtt ccacatacag gaacggccta 1560 cttcgcagat gagcccaccg aggctcaggc tccgggcgga ttctgcgtgt caccctcgct 1620 ccttggggtc cgctggccgg cctgtgccac ccggacgccc ggttcactgc ctctgtctcc 1680 cccatcagcg cagccccgga cgctatggcc cacccctcca gctggcccct cgagtaggat 1740 ggtagcacgt aaccaggtgg aagccgacaa tgcgatctcc ccggcatcag agccccgacg 1800 gcggccagag ccatcctcgt cctcgtcttc gtcctcgccg gcggccccgg cgcgtccccg 1860 gccctgcccg gtggtcccgg ccccggctcc gggcgacact cacttccgca ccttccgctc 1920 ccactctgat taccggcgca tcacgcggac cagcgctctc ctggacgcct gcggcttcta 1980 ctggggaccc ctgagcgtgc atggggcgca cgaacggctg cgttccgaac ccgtgggcac 2040 cttcttggtg cgcgacagtc gccagcggaa ctgcttcttc gcgctcagcg tgaagatggc 2100 ttcgggcccc acgagcattc gtgtgcactt ccaggccggc cgcttccacc tggacggcaa 2160 ccgcgagacc ttcgactgcc tcttcgagct gctggagcac tacgtggcgg cgccgcgccg 2220 catgttgggg gccccactgc gccagcgccg cgtgcggccg ctgcaggagc tgtgtcgcca 2280 gcgcatcgtg gccgccgtgg gtcgcgagaa cctggcacgc atccctctta acccggtact 2340 ccgtgactac ctgagttcct tccccttcca gatctgaccg gctgccgccg tgcccgcaga 2400 attaagtggg agcgccttat tatttcttat tattaattat tattattttt ctggaaccac 2460 gtgggagccc tccccgccta ggtcggaggg agtgggtgtg gagggtgaga tccctcccac 2520 ttctggctgg agaccttatc ccgcctctcg gggggcctcc cctcctggtg ctccctcccg 2580 gtccccctgg ttgtagcagc ttgtgtctgg ggccaggacc tgaactccac gcctacctct 2640 ccatgtttac atgttcccag tatctttgca caaaccaggg gtgggggagg gtctctggct 2700 tcatttttct gctgtgcaga atattctatt ttatattttt acatccagtt tagataataa 2760 actttattat gaaagttttt ttttttaaag aaacaaagat ttctaga 2807 <210> SEQ ID NO 12 <211> LENGTH: 212 <212> TYPE: PRT <213> ORGANISM: Rat <400> SEQUENCE: 12 Met Val Ala Arg Asn Gln Val Glu Ala Asp Asn Ala Ile Ser Pro Ala 1 5 10 15 Ser Glu Pro Arg Arg Arg Pro Glu Pro Ser Ser Ser Ser Ser Ser Ser 20 25 30 Ser Pro Ala Ala Pro Ala Arg Pro Arg Pro Cys Pro Val Val Pro Ala 35 40 45 Pro Ala Pro Gly Asp Thr His Phe Arg Thr Phe Arg Ser His Ser Asp 50 55 60 Tyr Arg Arg Ile Thr Arg Thr Ser Ala Leu Leu Asp Ala Cys Gly Phe 65 70 75 80 Tyr Trp Gly Pro Leu Ser Val His Gly Ala His Glu Arg Leu Arg Ser 85 90 95 Glu Pro Val Gly Thr Phe Leu Val Arg Asp Ser Arg Gln Arg Asn Cys 100 105 110 Phe Phe Ala Leu Ser Val Lys Met Ala Ser Gly Pro Thr Ser Ile Arg 115 120 125 Val His Phe Gln Ala Gly Arg Phe His Leu Asp Gly Asn Arg Glu Thr 130 135 140 Phe Asp Cys Leu Phe Glu Leu Leu Glu His Tyr Val Ala Ala Pro Arg 145 150 155 160 Arg Met Leu Gly Ala Pro Leu Arg Gln Arg Arg Val Arg Pro Leu Gln 165 170 175 Glu Leu Cys Arg Gln Arg Ile Val Ala Ala Val Gly Arg Glu Asn Leu 180 185 190 Ala Arg Ile Pro Leu Asn Pro Val Leu Arg Asp Tyr Leu Ser Ser Phe 195 200 205 Pro Phe Gln Ile 210 <210> SEQ ID NO 13 <211> LENGTH: 1611 <212> TYPE: DNA <213> ORGANISM: Mouse <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (263)..(1525) <400> SEQUENCE: 13 cgaattccgg gcgggctgtg tgagtctgtg agtggaaggc gcgccggctc ttttgtctga 60 gtgtgacccg gtggctttgt tccaggcatt ccggtgattt cctccgggca gtccgcagaa 120 gccgcagcgg ccgcccgcgc tctctctgca gtctccacac ccgggagagc ctgagcccgc 180 gtcacgcccc tcagcccccg ctgagtccct tctctgttgt cgcgtccgaa tcgagttccc 240 ggaatcagac ggtgccccat ag atg gcc agc ttt ccc ccg agg gtt aac gag 292 Met Ala Ser Phe Pro Pro Arg Val Asn Glu 1 5 10 aaa gag atc gtg aga tca cgt act ata ggg gaa ctc ttg gct cca gca 340 Lys Glu Ile Val Arg Ser Arg Thr Ile Gly Glu Leu Leu Ala Pro Ala 15 20 25 gct cct ttt gac aag aaa tgt ggt ggt gag aac tgg acg gtt gct ttt 388 Ala Pro Phe Asp Lys Lys Cys Gly Gly Glu Asn Trp Thr Val Ala Phe 30 35 40 gct cct gat ggt tcc tac ttt gcg tgg tca caa gga tat cgc ata gtg 436 Ala Pro Asp Gly Ser Tyr Phe Ala Trp Ser Gln Gly Tyr Arg Ile Val 45 50 55 aag ctt gtc ccg tgg tcc cag tgc cgt aag aac ttt ctt ttg cat ggt 484 Lys Leu Val Pro Trp Ser Gln Cys Arg Lys Asn Phe Leu Leu His Gly 60 65 70 tcc aaa aat gtt acc aat tca agc tgt cta aaa ttg gca aga caa aac 532 Ser Lys Asn Val Thr Asn Ser Ser Cys Leu Lys Leu Ala Arg Gln Asn 75 80 85 90 agt aat ggt ggt cag aaa aac aag cct cct gag cac gtt ata gac tgt 580 Ser Asn Gly Gly Gln Lys Asn Lys Pro Pro Glu His Val Ile Asp Cys 95 100 105 gga gac ata gtc tgg agt ctt gct ttt ggg tct tca gtt cca gaa aaa 628 Gly Asp Ile Val Trp Ser Leu Ala Phe Gly Ser Ser Val Pro Glu Lys 110 115 120 cag agt cgt tgc gtt aat ata gaa tgg cat cgg ttc cga ttt gga cag 676 Gln Ser Arg Cys Val Asn Ile Glu Trp His Arg Phe Arg Phe Gly Gln 125 130 135 gat cag cta ctc ctt gcc aca gga tta aac aat ggt cgc atc aaa atc 724 Asp Gln Leu Leu Leu Ala Thr Gly Leu Asn Asn Gly Arg Ile Lys Ile 140 145 150 tgg gat gta tat aca gga aaa ctc ctc ctt aat ttg gta gac cac att 772 Trp Asp Val Tyr Thr Gly Lys Leu Leu Leu Asn Leu Val Asp His Ile 155 160 165 170 gaa atg gtt aga gat tta act ttt gct cca gat ggg agc tta ctc ctt 820 Glu Met Val Arg Asp Leu Thr Phe Ala Pro Asp Gly Ser Leu Leu Leu 175 180 185 gta tca gct tca aga gac aaa act cta aga gtg tgg gac ctg aaa gat 868 Val Ser Ala Ser Arg Asp Lys Thr Leu Arg Val Trp Asp Leu Lys Asp 190 195 200 gat gga aac atg gtg aaa gta ttg cgg gca cat cag aat tgg gtg tac 916 Asp Gly Asn Met Val Lys Val Leu Arg Ala His Gln Asn Trp Val Tyr 205 210 215 agt tgt gca ttc tct ccc gac tgt tct atg ctg tgt tca gtg ggc gcc 964 Ser Cys Ala Phe Ser Pro Asp Cys Ser Met Leu Cys Ser Val Gly Ala 220 225 230 agt aaa gca gtt ttc ctt tgg aat atg gat aaa tac acc atg att agg 1012 Ser Lys Ala Val Phe Leu Trp Asn Met Asp Lys Tyr Thr Met Ile Arg 235 240 245 250 aag ctg gaa ggt cat cac cat gat gtt gta gct tgt gac ttt tct cct 1060 Lys Leu Glu Gly His His His Asp Val Val Ala Cys Asp Phe Ser Pro 255 260 265 gat gga gca ttg cta gct act gca tcc tat gac act cgt gtg tat gtc 1108 Asp Gly Ala Leu Leu Ala Thr Ala Ser Tyr Asp Thr Arg Val Tyr Val 270 275 280 tgg gat cca cac aat gga gac ctt ctg atg gag ttt ggg cac ctg ttt 1156 Trp Asp Pro His Asn Gly Asp Leu Leu Met Glu Phe Gly His Leu Phe 285 290 295 ccc tcg ccc act cca ata ttt gct gga gga gca aat gac cga tgg gtg 1204 Pro Ser Pro Thr Pro Ile Phe Ala Gly Gly Ala Asn Asp Arg Trp Val 300 305 310 aga gct gtg tct ttc agt cat gat gga ctg cat gtt gcc agc ctt gct 1252 Arg Ala Val Ser Phe Ser His Asp Gly Leu His Val Ala Ser Leu Ala 315 320 325 330 gat gat aaa atg gtg agg ttc tgg aga atc gat gag gat tgt ccg gta 1300 Asp Asp Lys Met Val Arg Phe Trp Arg Ile Asp Glu Asp Cys Pro Val 335 340 345 caa gtt gca cct ttg agc aat ggt ctt tgc tgt gcc ttt tct act gat 1348 Gln Val Ala Pro Leu Ser Asn Gly Leu Cys Cys Ala Phe Ser Thr Asp 350 355 360 ggc agt gtt tta gct gct ggg aca cat gat gga agt gtg tat ttt tgg 1396 Gly Ser Val Leu Ala Ala Gly Thr His Asp Gly Ser Val Tyr Phe Trp 365 370 375 gcc act cca agg caa gtc cct agc ctt caa cat ata tgt cgc atg tca 1444 Ala Thr Pro Arg Gln Val Pro Ser Leu Gln His Ile Cys Arg Met Ser 380 385 390 atc cga aga gtg atg tcc acc caa gaa gtc caa aaa ctg cct gtt cct 1492 Ile Arg Arg Val Met Ser Thr Gln Glu Val Gln Lys Leu Pro Val Pro 395 400 405 410 tcc aaa ata ttg gcg ttt ctc tcc tac cgc ggt tagactgaag actgcctttc 1545 Ser Lys Ile Leu Ala Phe Leu Ser Tyr Arg Gly 415 420 ctggtaggcc tgccagacag agcgcccttt acaagacaca cctcaagctt tacctcgtgc 1605 cgaatt 1611 <210> SEQ ID NO 14 <211> LENGTH: 421 <212> TYPE: PRT <213> ORGANISM: Mouse <400> SEQUENCE: 14 Met Ala Ser Phe Pro Pro Arg Val Asn Glu Lys Glu Ile Val Arg Ser 1 5 10 15 Arg Thr Ile Gly Glu Leu Leu Ala Pro Ala Ala Pro Phe Asp Lys Lys 20 25 30 Cys Gly Gly Glu Asn Trp Thr Val Ala Phe Ala Pro Asp Gly Ser Tyr 35 40 45 Phe Ala Trp Ser Gln Gly Tyr Arg Ile Val Lys Leu Val Pro Trp Ser 50 55 60 Gln Cys Arg Lys Asn Phe Leu Leu His Gly Ser Lys Asn Val Thr Asn 65 70 75 80 Ser Ser Cys Leu Lys Leu Ala Arg Gln Asn Ser Asn Gly Gly Gln Lys 85 90 95 Asn Lys Pro Pro Glu His Val Ile Asp Cys Gly Asp Ile Val Trp Ser 100 105 110 Leu Ala Phe Gly Ser Ser Val Pro Glu Lys Gln Ser Arg Cys Val Asn 115 120 125 Ile Glu Trp His Arg Phe Arg Phe Gly Gln Asp Gln Leu Leu Leu Ala 130 135 140 Thr Gly Leu Asn Asn Gly Arg Ile Lys Ile Trp Asp Val Tyr Thr Gly 145 150 155 160 Lys Leu Leu Leu Asn Leu Val Asp His Ile Glu Met Val Arg Asp Leu 165 170 175 Thr Phe Ala Pro Asp Gly Ser Leu Leu Leu Val Ser Ala Ser Arg Asp 180 185 190 Lys Thr Leu Arg Val Trp Asp Leu Lys Asp Asp Gly Asn Met Val Lys 195 200 205 Val Leu Arg Ala His Gln Asn Trp Val Tyr Ser Cys Ala Phe Ser Pro 210 215 220 Asp Cys Ser Met Leu Cys Ser Val Gly Ala Ser Lys Ala Val Phe Leu 225 230 235 240 Trp Asn Met Asp Lys Tyr Thr Met Ile Arg Lys Leu Glu Gly His His 245 250 255 His Asp Val Val Ala Cys Asp Phe Ser Pro Asp Gly Ala Leu Leu Ala 260 265 270 Thr Ala Ser Tyr Asp Thr Arg Val Tyr Val Trp Asp Pro His Asn Gly 275 280 285 Asp Leu Leu Met Glu Phe Gly His Leu Phe Pro Ser Pro Thr Pro Ile 290 295 300 Phe Ala Gly Gly Ala Asn Asp Arg Trp Val Arg Ala Val Ser Phe Ser 305 310 315 320 His Asp Gly Leu His Val Ala Ser Leu Ala Asp Asp Lys Met Val Arg 325 330 335 Phe Trp Arg Ile Asp Glu Asp Cys Pro Val Gln Val Ala Pro Leu Ser 340 345 350 Asn Gly Leu Cys Cys Ala Phe Ser Thr Asp Gly Ser Val Leu Ala Ala 355 360 365 Gly Thr His Asp Gly Ser Val Tyr Phe Trp Ala Thr Pro Arg Gln Val 370 375 380 Pro Ser Leu Gln His Ile Cys Arg Met Ser Ile Arg Arg Val Met Ser 385 390 395 400 Thr Gln Glu Val Gln Lys Leu Pro Val Pro Ser Lys Ile Leu Ala Phe 405 410 415 Leu Ser Tyr Arg Gly 420 <210> SEQ ID NO 15 <211> LENGTH: 783 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 15 ctgtcttcct ccgcagcgcg aggctgggta cagggtctat tgtctgtggt tgactccgta 60 ctttggtctg aggccttcgg gagctttccc gaggcagtta gcagaagccg cagcgaccgc 120 ccccgcccgt ctcctctgtc cctgggcccg ggagacaaac ttggcgtcac gccctcagcg 180 gtcgccactc tcttctctgt tgttgggtcc gcatcgtatt cccggaatca gacggtgccc 240 catagatggc cagctttccc ccgagggtca acgagaaaga gatcgtgaga tcacgtacta 300 taggtgaact tttagctcct gcagctcctt ttgacaagaa atgtggtcgt gaaaattgga 360 ctgttgcttt tgctccagat ggttcatact ttgcttggtc acaaggacat cgcacagtaa 420 agcttgttcc gtggtcccag tgccttcaga actttctctt gcatggcacc aagaatgtta 480 ccaattcaag cagtttaaga ttgccaagac aaaatagtga tggtggtcag aaaaataagc 540 ctcgtgacat attatagact gtggagatat agtctggagt cttgcttttg ggtcatcagt 600 tccagaaaaa cagagtcgct gtgtaaatat agaatggcat cgcttcagat ttggacaaga 660 tcagctactt cttgctacag ggttgaacaa tgggcgtatc aaaatatggg atgtatatca 720 ggaaactcct ccttaacttg gtagatcata ctgaagtggt cagagattta acttttgctc 780 cag 783 <210> SEQ ID NO 16 <211> LENGTH: 1122 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 16 ctctgtatgt ctgaatgaag ctataacatt tgccttttta ttgcaggttt tcctttggaa 60 tatggataaa tacaccatga tacggaaact agaaggacat caccatgatg tggtagcttg 120 tgacttttct cctgatggag cattactggc tactgcatct tatgatactc gagtatatat 180 ctgggatcca cataatggag acattctgat ggaatttggg cacctgtttc ccccacctac 240 tccaatattt gctggaggag caaatgaccg gtgggtacga tctgtatctt ttagccatga 300 tggactgcat gttgcaagcc ttgctgatga taaaatggtg aggttctgga gaattgatga 360 ggattatcca gtgcaagttg cacctttgag caatggtctt tgctgtgcct tctctactga 420 tggcagtgtt ttagctgctg ggacacatga cggaagtgtg tatttttggg ccactccacg 480 gcaggtccct agcctgcaac atttatgtcg catgtcaatc cgaagagtga tgcccaccca 540 agaagttcag gagctgccga ttccttccaa gcttttggag tttctctcgt atcgtattta 600 gaagattctg ccttccctag tagtagggac tgacagaata cacttaacac aaacctcaag 660 ctttactgac ttcaattatc tgtttttaaa gacgtagaag atttatttaa tttgatatgt 720 tcttgtactg cattttgatc agttgagctt ttaaaatatt atttatagac aatagaagta 780 tttctgaaca tatcaaatat aaattttttt aaagatctaa ctgtgaaaac atacatacct 840 gtacatattt agatataagc tgctatatgt tgaatggacc cttttgcttt tctgattttt 900 agttctgaca tgtatatatt gcttcagtag agccacaata tgtatctttg ctgtaaagtg 960 caaggaaatt ttaaattctg ggacactgag ttagatggta aatactgact tacgaaagtt 1020 gaattgggtg aggcgggcaa atcacctgag gtcagcagtt tgagactagc ctggcaaaca 1080 tgatgaaacc ctgtctctac taaaaataca aaaaaaaaaa aa 1122 <210> SEQ ID NO 17 <211> LENGTH: 2544 <212> TYPE: DNA <213> ORGANISM: Mouse <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (423)..(2030) <221> NAME/KEY: UNSURE <222> LOCATION: (320) <223> OTHER INFORMATION: Xaa is unsure <221> NAME/KEY: UNSURE <222> LOCATION: (451) <223> OTHER INFORMATION: Xaa is unsure <221> NAME/KEY: UNSURE <222> LOCATION: (531) <223> OTHER INFORMATION: Xaa is unsure <400> SEQUENCE: 17 cggcacgagc cgggctccgt ccggaggaag cgaggctgcg ccgccggccc ggcaggagcg 60 gaggacggga mgcgcgggcg gtcgcgctcg ccctgtcgct gactgcgctg ccccggccca 120 tccttgcctg gccgcaggtg ccctggatga ggccgccgcg cgtgtcccgg ccgctgagtg 180 tcccccgcgg tcgcccggcg cctgccctca agcggccgcc tctccttgcc cgggtccccg 240 ttttcccccg gcgcagtcct cctccggtgg gcgcctccgc acctcggcgc aggcggcacg 300 gccctcgggc cgggatggat ccgccgggaa gaggaagaca agccggggcg ttgagcccct 360 gcgcacggtg ccgccgcgcg tagtgggagc ttactcgcag taggctctcg ctcttctaat 420 ca atg gat aaa gtg ggg aaa atg tgg aac aac tta aaa tac aga tgc 467 Met Asp Lys Val Gly Lys Met Trp Asn Asn Leu Lys Tyr Arg Cys 1 5 10 15 cag aat ctc ttc agc cac gag gga gga agc cgt aat gag aac gtg gag 515 Gln Asn Leu Phe Ser His Glu Gly Gly Ser Arg Asn Glu Asn Val Glu 20 25 30 atg aac ccc aac aga tgt ccg tct gtc aaa gag aaa agc atc agt ctg 563 Met Asn Pro Asn Arg Cys Pro Ser Val Lys Glu Lys Ser Ile Ser Leu 35 40 45 gga gag gca gct ccc cag caa gag agc agt ccc tta aga gaa aat gtt 611 Gly Glu Ala Ala Pro Gln Gln Glu Ser Ser Pro Leu Arg Glu Asn Val 50 55 60 gcc tta cag ctg gga ctg agc cct tcc aag acc ttt tcc agg cgg aac 659 Ala Leu Gln Leu Gly Leu Ser Pro Ser Lys Thr Phe Ser Arg Arg Asn 65 70 75 caa aac tgt gcc gca gag atc cct caa gtg gtt gaa atc agc atc gag 707 Gln Asn Cys Ala Ala Glu Ile Pro Gln Val Val Glu Ile Ser Ile Glu 80 85 90 95 aaa gac agt gac tcg ggt gcc acc cca gga acg agg ctt gca cgg aga 755 Lys Asp Ser Asp Ser Gly Ala Thr Pro Gly Thr Arg Leu Ala Arg Arg 100 105 110 gac tcc tac tcg cgg cac gcc ccg tgg gga gga aag aag aaa cat tcc 803 Asp Ser Tyr Ser Arg His Ala Pro Trp Gly Gly Lys Lys Lys His Ser 115 120 125 tgt tcc aca aag acc cag agt tca ttg gat acc gag aaa aag ttt ggt 851 Cys Ser Thr Lys Thr Gln Ser Ser Leu Asp Thr Glu Lys Lys Phe Gly 130 135 140 aga act cga agc ggc ctt cag agg cga gag cgg cgc tat gga gtc agc 899 Arg Thr Arg Ser Gly Leu Gln Arg Arg Glu Arg Arg Tyr Gly Val Ser 145 150 155 tcc atg cag gac atg gac agc gtt tct agc cgc gcg gtc ggg agc cgc 947 Ser Met Gln Asp Met Asp Ser Val Ser Ser Arg Ala Val Gly Ser Arg 160 165 170 175 tcc ctg agg cag agg ctc cag gac acg gtg ggt ttg tgt ttt ccc atg 995 Ser Leu Arg Gln Arg Leu Gln Asp Thr Val Gly Leu Cys Phe Pro Met 180 185 190 aga act tac agc aag cag tca aag cca ctc ttt tcc aat aaa aga aaa 1043 Arg Thr Tyr Ser Lys Gln Ser Lys Pro Leu Phe Ser Asn Lys Arg Lys 195 200 205 ata cat ctt tct gaa tta atg ctg gag aaa tgc cct ttt cct gct ggc 1091 Ile His Leu Ser Glu Leu Met Leu Glu Lys Cys Pro Phe Pro Ala Gly 210 215 220 tcg gat tta gca caa aag tgg cat ttg att aaa cag cat acc gcc cct 1139 Ser Asp Leu Ala Gln Lys Trp His Leu Ile Lys Gln His Thr Ala Pro 225 230 235 gtg agc cca cac tca aca ttt ttt gat aca ttt gat cca tca ctg gtg 1187 Val Ser Pro His Ser Thr Phe Phe Asp Thr Phe Asp Pro Ser Leu Val 240 245 250 255 tct aca gaa gat gaa gaa gat agg ctt cgc gag aga aga cgg ctt agt 1235 Ser Thr Glu Asp Glu Glu Asp Arg Leu Arg Glu Arg Arg Arg Leu Ser 260 265 270 atc gaa gaa ggg gtg gat ccc cct ccc aac gca caa ata cac acc ttt 1283 Ile Glu Glu Gly Val Asp Pro Pro Pro Asn Ala Gln Ile His Thr Phe gaa gct act gca cag gtc aac cca ttg tat aag ctg gga cca aag tta 1331 Glu Ala Thr Ala Gln Val Asn Pro Leu Tyr Lys Leu Gly Pro Lys Leu 290 295 300 gct cct ggg atg aca gag ata agt gga gat ggt tct gca att cca caa 1379 Ala Pro Gly Met Thr Glu Ile Ser Gly Asp Gly Ser Ala Ile Pro Gln 305 310 315 gcs aat tgt gac tca gaa gag gat tca acc acc cta tgt ctg cag tca 1427 Xaa Asn Cys Asp Ser Glu Glu Asp Ser Thr Thr Leu Cys Leu Gln Ser 320 325 330 335 cgg agg cag aag cag cgc cag gtg tcc ggg gac agc cac gcg cac gtt 1475 Arg Arg Gln Lys Gln Arg Gln Val Ser Gly Asp Ser His Ala His Val 340 345 350 agc aga cag gga gct tgg aaa gtt cat acg cag atc gat tac ata cac 1523 Ser Arg Gln Gly Ala Trp Lys Val His Thr Gln Ile Asp Tyr Ile His 355 360 365 tgc ctc gtg cca gat ttg ctt cag atc aca ggg aat ccc tgt tac tgg 1571 Cys Leu Val Pro Asp Leu Leu Gln Ile Thr Gly Asn Pro Cys Tyr Trp 370 375 380 ggc gtg atg gac cga tac gag gcc gaa gcc ctt cta gaa ggg aaa ccg 1619 Gly Val Met Asp Arg Tyr Glu Ala Glu Ala Leu Leu Glu Gly Lys Pro 385 390 395 gaa ggc acg ttc ttg ctc agg gac tct gca cag gag gac tac ctc ttc 1667 Glu Gly Thr Phe Leu Leu Arg Asp Ser Ala Gln Glu Asp Tyr Leu Phe 400 405 410 415 tct gtg agc ttc cgc cgc tac aac agg tct ctg cac gcc cgg atc gag 1715 Ser Val Ser Phe Arg Arg Tyr Asn Arg Ser Leu His Ala Arg Ile Glu 420 425 430 cag tgg aac cac aac ttc agc ttc gat gcc cat gac ccc tgc gtg ttt 1763 Gln Trp Asn His Asn Phe Ser Phe Asp Ala His Asp Pro Cys Val Phe 435 440 445 cac tcc tcc acw gtc acg ggg ctt ctc gaa cac tat aaa gac ccc agc 1811 His Ser Ser Xaa Val Thr Gly Leu Leu Glu His Tyr Lys Asp Pro Ser 450 455 460 tct tgc atg ttt ttt gaa ccg ttg cta acg ata tca ctg aat aga act 1859 Ser Cys Met Phe Phe Glu Pro Leu Leu Thr Ile Ser Leu Asn Arg Thr 465 470 475 ttc cct ttc agc ctg cag tat atc tgc cgc gca gtg atc tgc aga tgc 1907 Phe Pro Phe Ser Leu Gln Tyr Ile Cys Arg Ala Val Ile Cys Arg Cys 480 485 490 495 act acg tat gat ggg att gac ggg ctc ccg cta ccg tcg atg tta cag 1955 Thr Thr Tyr Asp Gly Ile Asp Gly Leu Pro Leu Pro Ser Met Leu Gln 500 505 510 gat ttt tta aaa gag tat cat tat aaa caa aaa gtt agg gtt cgc tgg 2003 Asp Phe Leu Lys Glu Tyr His Tyr Lys Gln Lys Val Arg Val Arg Trp 515 520 525 tta gaa cga gar cca gtc aaa gca aag taactcctgt ccccaaaggg 2050 Leu Glu Arg Xaa Pro Val Lys Ala Lys 530 535 cactaactaa gtctgctcct cccgtgcatc mgaactgcac ccataggrag gcagtcagct 2110 gctaggattt cccacccaga atgggagctt agtcattagc ctctgcccta tggggtccgc 2170 tgttcctcag acaaaggtgc ctagggacag caagatggct tgcaggtgtt cggtgggctg 2230 tgacaactga gggaggcaac tctggggcat ttgctatgaa gaattctatt tcttaccgaa 2290 gaacaaatta ttaatattgg atgggtattt caatagtgtg actaatgttt gaaattattt 2350 tttctaagaa tttttctata accttcagaa aaagtagtga tgtttgtagt tactataaat 2410 caagctttga aagttcaaaa caaacaagtt aaataaaaga ctaccttcct tttagagaaa 2470 acaaatgcaa gttttcccag ccacaggcat tgtgcactgt taatgttagc ttgttatcag 2530 ctcctttctc ctcc 2544 <210> SEQ ID NO 18 <211> LENGTH: 536 <212> TYPE: PRT <213> ORGANISM: Mouse <220> FEATURE: <221> NAME/KEY: UNSURE <222> LOCATION: (320) <223> OTHER INFORMATION: Xaa is unsure <221> NAME/KEY: UNSURE <222> LOCATION: (451) <223> OTHER INFORMATION: Xaa is unsure <221> NAME/KEY: UNSURE <222> LOCATION: (531) <223> OTHER INFORMATION: Xaa is unsure <400> SEQUENCE: 18 Met Asp Lys Val Gly Lys Met Trp Asn Asn Leu Lys Tyr Arg Cys Gln 1 5 10 15 Asn Leu Phe Ser His Glu Gly Gly Ser Arg Asn Glu Asn Val Glu Met 20 25 30 Asn Pro Asn Arg Cys Pro Ser Val Lys Glu Lys Ser Ile Ser Leu Gly 35 40 45 Glu Ala Ala Pro Gln Gln Glu Ser Ser Pro Leu Arg Glu Asn Val Ala 50 55 60 Leu Gln Leu Gly Leu Ser Pro Ser Lys Thr Phe Ser Arg Arg Asn Gln 65 70 75 80 Asn Cys Ala Ala Glu Ile Pro Gln Val Val Glu Ile Ser Ile Glu Lys 85 90 95 Asp Ser Asp Ser Gly Ala Thr Pro Gly Thr Arg Leu Ala Arg Arg Asp 100 105 110 Ser Tyr Ser Arg His Ala Pro Trp Gly Gly Lys Lys Lys His Ser Cys 115 120 125 Ser Thr Lys Thr Gln Ser Ser Leu Asp Thr Glu Lys Lys Phe Gly Arg 130 135 140 Thr Arg Ser Gly Leu Gln Arg Arg Glu Arg Arg Tyr Gly Val Ser Ser 145 150 155 160 Met Gln Asp Met Asp Ser Val Ser Ser Arg Ala Val Gly Ser Arg Ser 165 170 175 Leu Arg Gln Arg Leu Gln Asp Thr Val Gly Leu Cys Phe Pro Met Arg 180 185 190 Thr Tyr Ser Lys Gln Ser Lys Pro Leu Phe Ser Asn Lys Arg Lys Ile 195 200 205 His Leu Ser Glu Leu Met Leu Glu Lys Cys Pro Phe Pro Ala Gly Ser 210 215 220 Asp Leu Ala Gln Lys Trp His Leu Ile Lys Gln His Thr Ala Pro Val 225 230 235 240 Ser Pro His Ser Thr Phe Phe Asp Thr Phe Asp Pro Ser Leu Val Ser 245 250 255 Thr Glu Asp Glu Glu Asp Arg Leu Arg Glu Arg Arg Arg Leu Ser Ile 260 265 270 Glu Glu Gly Val Asp Pro Pro Pro Asn Ala Gln Ile His Thr Phe Glu 275 280 285 Ala Thr Ala Gln Val Asn Pro Leu Tyr Lys Leu Gly Pro Lys Leu Ala 290 295 300 Pro Gly Met Thr Glu Ile Ser Gly Asp Gly Ser Ala Ile Pro Gln Xaa 305 310 315 320 Asn Cys Asp Ser Glu Glu Asp Ser Thr Thr Leu Cys Leu Gln Ser Arg 325 330 335 Arg Gln Lys Gln Arg Gln Val Ser Gly Asp Ser His Ala His Val Ser 340 345 350 Arg Gln Gly Ala Trp Lys Val His Thr Gln Ile Asp Tyr Ile His Cys 355 360 365 Leu Val Pro Asp Leu Leu Gln Ile Thr Gly Asn Pro Cys Tyr Trp Gly 370 375 380 Val Met Asp Arg Tyr Glu Ala Glu Ala Leu Leu Glu Gly Lys Pro Glu 385 390 395 400 Gly Thr Phe Leu Leu Arg Asp Ser Ala Gln Glu Asp Tyr Leu Phe Ser 405 410 415 Val Ser Phe Arg Arg Tyr Asn Arg Ser Leu His Ala Arg Ile Glu Gln 420 425 430 Trp Asn His Asn Phe Ser Phe Asp Ala His Asp Pro Cys Val Phe His 435 440 445 Ser Ser Xaa Val Thr Gly Leu Leu Glu His Tyr Lys Asp Pro Ser Ser 450 455 460 Cys Met Phe Phe Glu Pro Leu Leu Thr Ile Ser Leu Asn Arg Thr Phe 465 470 475 480 Pro Phe Ser Leu Gln Tyr Ile Cys Arg Ala Val Ile Cys Arg Cys Thr 485 490 495 Thr Tyr Asp Gly Ile Asp Gly Leu Pro Leu Pro Ser Met Leu Gln Asp 500 505 510 Phe Leu Lys Glu Tyr His Tyr Lys Gln Lys Val Arg Val Arg Trp Leu 515 520 525 Glu Arg Xaa Pro Val Lys Ala Lys 530 535 <210> SEQ ID NO 19 <211> LENGTH: 1221 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 19 gattaaacag catacagctc ctgtgagccc acattcaaca ttttttgata ctttgatcca 60 tctttggttt ctacagaaga tgaagaagat aggcttagag agagaaggcg gcttagtatt 120 gaagaagggg ttgatccccc tcccaatgca caaatacata catttgaagc tactgcacag 180 gttaatccat tattaaactg ggaccaaaat tagctcctgg aatgactgaa ataagtgggg 240 acagttctgc aattccacaa gctaattgtg actcggaaga ggatacaacc accctgtgtt 300 gcagtcacgg aggcagaagc agcgtcagat atctggagac agccataccc atgttagcag 360 acagggagct tggaaagtcc acacacagat tgattacata cactgcttcg tgcctgattt 420 gcttcaaatt acagggaatc cctgttactg gggagtgatg gaccgttatg aagcagaagc 480 ccttctcgaa gggaaacctg aaggcacgtt tttgctcagg gactctgcgc aagaggacta 540 cttcttctct gtgagcttcc gccgatacaa cagatccctg catgcccgaa ttgagcagtg 600 gaatcacaac tttagtttcg acgcccatga cccgtgtgta tttcactcct ccactgtaac 660 gggactttta gaacattata aagatcccag ttcgtgcatg ttttttgaac cattgcttac 720 tatatcacta aataggactt tcccttttag cctgcagtat atctgtcgcg cggtaatctg 780 caggtgcact acgtatgatg gaattgatgg gctccctcta ccctcaatgt tacaggattt 840 tttaaaagag tatcattata aacaaaaagt tagagttcgc tggttggaac gagaaccagt 900 caaggcaaag taaactctcc ggtccccaaa gggtgttaac taggtccgct ttcatgtgca 960 tcagacagta cacctatagc aagcacacgt agcagtgtta ggctttttca tacagtatgt 1020 aagcttagtg ttagtatctg tcagatgcta cctgctgtta cttattcaga taaacatggt 1080 gcctattgga acaatagcgg atagagctac aggtgttcag taagactaca aaaacatttt 1140 gcctatttcg ctaacagttt ggtttttaat ggctgtggta tttgagtgag gcaactctgg 1200 ggcatttgtt atgaagaaat g 1221 <210> SEQ ID NO 20 <211> LENGTH: 2369 <212> TYPE: DNA <213> ORGANISM: Murine <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (116)..(1327) <400> SEQUENCE: 20 ggcacgaggc ggtggtggcg gcggcgggcg cggccgcggc ggggcgggcg cggaatgaag 60 gcccacggcc ctgggggctg aggcgcccgc cgcctggggc gggccgcgcg tcctc atg 118 Met 1 gag gcc gga gag gag ccg ctg ctg ctg gct gaa ctc aag cct ggg cgc 166 Glu Ala Gly Glu Glu Pro Leu Leu Leu Ala Glu Leu Lys Pro Gly Arg 5 10 15 ccc cac cag ttc gac tgg aag tca agc tgc gag acc tgg agc gtg gcc 214 Pro His Gln Phe Asp Trp Lys Ser Ser Cys Glu Thr Trp Ser Val Ala 20 25 30 ttc tcg cca gac ggt tcc tgg ttc gcc tgg tct caa gga cac tgc gtg 262 Phe Ser Pro Asp Gly Ser Trp Phe Ala Trp Ser Gln Gly His Cys Val 35 40 45 gtc aag ctg gtc ccc tgg ccc tta gag gaa cag ttc atc cct aaa gga 310 Val Lys Leu Val Pro Trp Pro Leu Glu Glu Gln Phe Ile Pro Lys Gly 50 55 60 65 ttc gaa gcc aag agc cga agc agc aag aat gac cca aaa gga cgg ggc 358 Phe Glu Ala Lys Ser Arg Ser Ser Lys Asn Asp Pro Lys Gly Arg Gly 70 75 80 agt ctg aag gag aag acg ctg gac tgt ggc cag att gtg tgg ggg ctg 406 Ser Leu Lys Glu Lys Thr Leu Asp Cys Gly Gln Ile Val Trp Gly Leu 85 90 95 gcc ttc agc ccg tgg ccc tct cca ccc agc agg aaa ctc tgg gca cgt 454 Ala Phe Ser Pro Trp Pro Ser Pro Pro Ser Arg Lys Leu Trp Ala Arg 100 105 110 cac cat ccc cag gcg cct gat gtt tct tgc ctg atc ctg gcc aca ggt 502 His His Pro Gln Ala Pro Asp Val Ser Cys Leu Ile Leu Ala Thr Gly 115 120 125 ctc aac gat ggg cag atc aag att tgg gag gta cag aca ggc ctc ctg 550 Leu Asn Asp Gly Gln Ile Lys Ile Trp Glu Val Gln Thr Gly Leu Leu 130 135 140 145 ctt ctg aat ctt tct ggc cac caa gac gtc gtg aga gat ctg agc ttc 598 Leu Leu Asn Leu Ser Gly His Gln Asp Val Val Arg Asp Leu Ser Phe 150 155 160 acg ccc agc ggc agt ttg att ttg gtc tct gca tcc cgg gat aag aca 646 Thr Pro Ser Gly Ser Leu Ile Leu Val Ser Ala Ser Arg Asp Lys Thr 165 170 175 ctt cga att tgg gac ctg aat aaa cac ggt aag cag atc cag gtg tta 694 Leu Arg Ile Trp Asp Leu Asn Lys His Gly Lys Gln Ile Gln Val Leu 180 185 190 tcc ggc cat ctg cag tgg gtt tac tgc tgc tcc atc tcc cct gac tgt 742 Ser Gly His Leu Gln Trp Val Tyr Cys Cys Ser Ile Ser Pro Asp Cys 195 200 205 agc atg ctg tgc tct gca gct ggg gag aag tcg gtc ttt ctg tgg agc 790 Ser Met Leu Cys Ser Ala Ala Gly Glu Lys Ser Val Phe Leu Trp Ser 210 215 220 225 atg cgg tcc tac aca cta atc cgg aaa cta gaa ggc cac caa agc agt 838 Met Arg Ser Tyr Thr Leu Ile Arg Lys Leu Glu Gly His Gln Ser Ser 230 235 240 gtt gtc tcc tgt gat ttc tct cct gat tca gcc ttg ctt gtc aca gct 886 Val Val Ser Cys Asp Phe Ser Pro Asp Ser Ala Leu Leu Val Thr Ala 245 250 255 tcg tat gac acc agt gtg att atg tgg gac ccc tac acc ggc gcg agg 934 Ser Tyr Asp Thr Ser Val Ile Met Trp Asp Pro Tyr Thr Gly Ala Arg 260 265 270 ctg agg tca ctt cat cac aca caa ctt gaa ccc acc atg gat gac agt 982 Leu Arg Ser Leu His His Thr Gln Leu Glu Pro Thr Met Asp Asp Ser 275 280 285 gac gtc cac atg agc tcc ctg agg tcc gtg tgc ttc tca cct gaa ggc 1030 Asp Val His Met Ser Ser Leu Arg Ser Val Cys Phe Ser Pro Glu Gly 290 295 300 305 ttg tat ctc gct acg gtg gca gat gac agg ctg ctc agg atc tgg gct 1078 Leu Tyr Leu Ala Thr Val Ala Asp Asp Arg Leu Leu Arg Ile Trp Ala 310 315 320 ctg gaa ctg aag gct ccg gtt gcc ttt gct ccg atg acc aat ggt ctt 1126 Leu Glu Leu Lys Ala Pro Val Ala Phe Ala Pro Met Thr Asn Gly Leu 325 330 335 tgc tgc acg ttc ttc cca cac ggt gga att att gcc aca ggg acg aga 1174 Cys Cys Thr Phe Phe Pro His Gly Gly Ile Ile Ala Thr Gly Thr Arg 340 345 350 gat ggc cat gtc cag ttc tgg aca gct ccc cgg gtc ctg tcc tca ctg 1222 Asp Gly His Val Gln Phe Trp Thr Ala Pro Arg Val Leu Ser Ser Leu 355 360 365 aag cac tta tgc agg aaa gcc ctc cga agt ttc ctg aca acg tat caa 1270 Lys His Leu Cys Arg Lys Ala Leu Arg Ser Phe Leu Thr Thr Tyr Gln 370 375 380 385 gtc cta gca ctg cca atc ccc aag aag atg aaa gag ttc ctc aca tac 1318 Val Leu Ala Leu Pro Ile Pro Lys Lys Met Lys Glu Phe Leu Thr Tyr 390 395 400 agg act ttc tagcagtgcc ggctccccca cctcctgcag cagcagcagt 1367 Arg Thr Phe acaagggact ggctaggatg gagtcaggca gctcacactg gaccagtgtg gaccttcctt 1427 cctcccatgg catgtgcaag taggtctgcg tgaccccact tctgtggtgc cggccttacc 1487 tcgtcttcat ccgtggtgag cagccttcgt cagtctagtt gtgttgaagc caagtgcagt 1547 tgtggatgtt gctggggtaa taaaggcaag cgggctccag agcctctctg gtggcggcca 1607 agccacactc ccttaactgg gaagtacctg ccacgtaggg catttctgct gcctatttcc 1667 agccagcggc tgcatggttt gaagttcctc cgttgtggtc agaagaactc tggtgtttgg 1727 ttccctgctc agctgcgcgt ggactgggct gagctcctca ccatacacta gtgccggctt 1787 ttgtttcctg taaacagtgg ttgcatgtgt agagaagtaa caagcgagta ttcagatcat 1847 acgaggaggc gttcctcggt gcatgacggt cagatggcca tttatcagca tatttatttg 1907 tattttctca gcacatagta aggtacaact gtgttttctc aattgtctcg aaaaaacaga 1967 gttcttaagt ggcccagttg tggagccaag tctaagtcgt gtggagtcag tgctgacatc 2027 actggcttgt gctgtctgtc acatgtgttt gtctctgctg cttgacctca tgggatgtac 2087 cctccagttc aactgcccaa aacagacagc cccttccaag caccgttctt tgacagcggt 2147 agcagctacc tattcaagac gcctcacaca aaatctgcct tagaaagtta atatatttta 2207 aattatttta aaagaaactc aacatcttat tctttggcct ttcttaattg atgctttatg 2267 gaggcagtgt taacattgta cagtgtatgc atagaggagt ctcctctatt tgaagaacaa 2327 tgcaaaatga ggctttcatt gaagggaaaa aaaaaaaaaa aa 2369 <210> SEQ ID NO 21 <211> LENGTH: 404 <212> TYPE: PRT <213> ORGANISM: Murine <400> SEQUENCE: 21 Met Glu Ala Gly Glu Glu Pro Leu Leu Leu Ala Glu Leu Lys Pro Gly 1 5 10 15 Arg Pro His Gln Phe Asp Trp Lys Ser Ser Cys Glu Thr Trp Ser Val 20 25 30 Ala Phe Ser Pro Asp Gly Ser Trp Phe Ala Trp Ser Gln Gly His Cys 35 40 45 Val Val Lys Leu Val Pro Trp Pro Leu Glu Glu Gln Phe Ile Pro Lys 50 55 60 Gly Phe Glu Ala Lys Ser Arg Ser Ser Lys Asn Asp Pro Lys Gly Arg 65 70 75 80 Gly Ser Leu Lys Glu Lys Thr Leu Asp Cys Gly Gln Ile Val Trp Gly 85 90 95 Leu Ala Phe Ser Pro Trp Pro Ser Pro Pro Ser Arg Lys Leu Trp Ala 100 105 110 Arg His His Pro Gln Ala Pro Asp Val Ser Cys Leu Ile Leu Ala Thr 115 120 125 Gly Leu Asn Asp Gly Gln Ile Lys Ile Trp Glu Val Gln Thr Gly Leu 130 135 140 Leu Leu Leu Asn Leu Ser Gly His Gln Asp Val Val Arg Asp Leu Ser 145 150 155 160 Phe Thr Pro Ser Gly Ser Leu Ile Leu Val Ser Ala Ser Arg Asp Lys 165 170 175 Thr Leu Arg Ile Trp Asp Leu Asn Lys His Gly Lys Gln Ile Gln Val 180 185 190 Leu Ser Gly His Leu Gln Trp Val Tyr Cys Cys Ser Ile Ser Pro Asp 195 200 205 Cys Ser Met Leu Cys Ser Ala Ala Gly Glu Lys Ser Val Phe Leu Trp 210 215 220 Ser Met Arg Ser Tyr Thr Leu Ile Arg Lys Leu Glu Gly His Gln Ser 225 230 235 240 Ser Val Val Ser Cys Asp Phe Ser Pro Asp Ser Ala Leu Leu Val Thr 245 250 255 Ala Ser Tyr Asp Thr Ser Val Ile Met Trp Asp Pro Tyr Thr Gly Ala 260 265 270 Arg Leu Arg Ser Leu His His Thr Gln Leu Glu Pro Thr Met Asp Asp 275 280 285 Ser Asp Val His Met Ser Ser Leu Arg Ser Val Cys Phe Ser Pro Glu 290 295 300 Gly Leu Tyr Leu Ala Thr Val Ala Asp Asp Arg Leu Leu Arg Ile Trp 305 310 315 320 Ala Leu Glu Leu Lys Ala Pro Val Ala Phe Ala Pro Met Thr Asn Gly 325 330 335 Leu Cys Cys Thr Phe Phe Pro His Gly Gly Ile Ile Ala Thr Gly Thr 340 345 350 Arg Asp Gly His Val Gln Phe Trp Thr Ala Pro Arg Val Leu Ser Ser 355 360 365 Leu Lys His Leu Cys Arg Lys Ala Leu Arg Ser Phe Leu Thr Thr Tyr 370 375 380 Gln Val Leu Ala Leu Pro Ile Pro Lys Lys Met Lys Glu Phe Leu Thr 385 390 395 400 Tyr Arg Thr Phe <210> SEQ ID NO 22 <211> LENGTH: 1246 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 22 gacactgcat cgtcaaactg atcccctggc cgttggagga gcagttcatc cctaaagggt 60 ttgaagccaa aagccgaagt agcaaaaatg agacgaaagg gcggggcagc ccaaaagaga 120 agacgctgga ctgtggtcag attgtctggg ggctggcctt cagcctgtgc tttccccacc 180 cagcaggaag ctctgggcac gccaccaccc ccaagtgccc gatgtctctt gcctggttct 240 tgctacggga ctcaacgatg ggcagatcaa gatctgggag gtgcagacag ggctcctgct 300 tttgaatctt tccggccacc aagatgtcgt gagagatctg agcttcacac ccagtggcag 360 tttgattttg gtctccgcgt cacgggataa gactcttcgc atctgggacc tgaataaaca 420 cggtaaacag attcaagtgt tatcgggcca cctgcagtgg gtttactgct gttccatctc 480 cccagactgc agcatgctgt gctctgcagc tggagagaag tcggtctttc tatggagcat 540 gaggtcctac acgttaattc ggaagctaga gggccatcaa agcagtgttg tctcttgtga 600 cttctccccc gactctgccc tgcttgtcac ggcttcttac gataccaatg tgattatgtg 660 ggacccctac accggcgaaa ggctgaggtc actccaccac acccaggttg accccgccat 720 ggatgacagt gacgtccaca ttagctcact gagatctgtg tgcttctctc cagaaggctt 780 gtaccttgcc acggtggcag atgacagact cctcaggatc tgggccctgg aactgaaaac 840 tcccattgca tttgctccta tgaccaatgg gctttgctgg cacatttttt ccacatggtg 900 gagtcattgc cacagggaca agagatggcc acgtccagtt ctggacagct cctagggtcc 960 tgtcctcact gaagcactta tgccggaaag cccttcgaag tttcctaaca acttaccaag 1020 tcctagcact gccaatcccc aagaaaatga aagagttcct cacatacagg actttttaag 1080 caacaccaca tcttgtgctt ctttgtagca gggtaaatcg tcctgtcaaa gggagttgct 1140 ggaataatgg gccaaacatc tggtcttgca ttgaaatagc atttctttgg gattgtgaat 1200 agaatgtagc aaaaccagat tccagtgtac tagtcatgga tttttc 1246 <210> SEQ ID NO 23 <211> LENGTH: 422 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 23 accatggttc caagtcctct cccctgtggt caagttgccc gaatgttggg cccaagtgcc 60 ttttcctcct tgggcctccc cttctgacct gcaggacagt tttccggagc ccatttggta 120 tgaggtatta attagcctta actaaattac aggggactca gaggccgtgc tcctgaccga 180 tccagacact attttttttt ttttttttta acaatggtgt gcatgtgcag gaaatgacaa 240 atttgtatgt cagattatac aaggatgtat tcttaaaccg catgactatt cagatggcta 300 ctgagttatc agtggccatt tattagcatc atatttattt gtattttctc aacagatgtt 360 aaggtacaac tgtgtttttc tcgattatct aaaaaccata gtacttaaat tgaaaaaaaa 420 aa 422 <210> SEQ ID NO 24 <211> LENGTH: 2019 <212> TYPE: DNA <213> ORGANISM: Mouse <220> FEATURE: <221> NAME/KEY: UNSURE <222> LOCATION: (2000) <223> OTHER INFORMATION: n is unsure <400> SEQUENCE: 24 ggcacgaggc ggggtcaggg cggaggctga ggaccaagta ggcatggcgg agggcgggac 60 cggccccgat ggacgggccg gcccgggacc cgcaggtcct aatctgaagg agtggctgag 120 ggagcagttc tgtgaccatc cactggagca ctgtgacgat acaagactcc atgatgcagc 180 ctatgtaggg gacctccaga ccctcaggaa cctactgcaa gaggagagct accggagccg 240 catcaatgag aagtctgtct ggtgctgcgg ctggcttccc tgcacaccac tgaggatcgc 300 agccactgca ggccatggga actgtgtgga cttcctcata cgcaaagggg ccgaggtgga 360 cctggtggat gtcaaggggc agactgccct gtatgtggct gtagtgaacg ggcacttgga 420 gagcactgag atccttttgg aagctggtgc tgatcccaac ggcagccggc accaccgcag 480 cactcctgtg taccatgcct ytcgtgtggg tagggacgac atcctgaagg ctcttatcag 540 gtatggggca gatgttgatg tcaaccatca tctgaattct gacacccggc cccctttttc 600 acggcggcta acctccttgg tggtctgtcc tctatacatc agtgctgcct accataacct 660 tcagtgcttc aggctgctct tgcaggctgg ggcaaatcct gacttcaatt gcaatggccc 720 tgtcaacacc caggagttct acaggggatc ccctgggtgt gtcatggatg ctgtcctgcg 780 ccatggctgt gaagcagcct tcgtgagtct gttggtagag tttggagcca acctgaacct 840 ggtgaagtgg gaatccctgg gcccagaggc aagaggcaga agaaagatgg atcctgaggc 900 cttgcaggtc tttaaagagg ccagaagtat tcccaggacc ttgctgagtt tgtgccgggt 960 ggctgtgaga agagctcttg gcaaataccg actgcatctg gttccctcgc tgccgctgcc 1020 agaccccata aagaagtttt tgctttatga gtagcattca catgcagtgc tgactgcaat 1080 gtggaagccg atcacctgca gtgaaaactg acacagactc tggcatcctg ggaaccatgg 1140 cctgtgctgc cagcttgatc cttggctgtc agtgaagaaa aaacggctgt gttctcttgg 1200 actgtgattc tatctcaggt gcttgggcca tcgaacgctc cttgagtcat tgtcaactga 1260 gaggcacata caaacttaat tttgttcctc ttcagtctct ctgttttgga ttcttcctgg 1320 caatgtgtgc agcatgggct gagcctggtg attgccctag tggggaaggc ttttttctcc 1380 aggctatgca tctatttatg ttcctacttt gcaatttatt gttcttttaa ggcttgatat 1440 caaaacagaa agaggtttgt taagaaaaga tatagggaga aaggaattcc ggttccgtgc 1500 acttgctagc ctgctttcct tgcctgggtt tgtctgtcta tgctgcctgg tgcacatccc 1560 ttctctttgc tgccactgtt ctattttggg agttgtcttc cgtctaagat ggcttctggg 1620 gttctatctt attgcacaga ggtcccagaa cagtgttcat agggcaccat ctgctctgcc 1680 aagggttttc tgatgtctta ccctggggat cttcagacag tggttacctt taggagaccc 1740 acctggaact aaccattaag tgactgccca cattcagatc agggaccatc ttaatagtac 1800 tcactgccag tcctcacaag agaagatgac acgggtgctc tcttcagaca ctcccataca 1860 ggaagttgga aaatgtcttg gtcacctggg ttgttcccag gctacaactt cttggtgttc 1920 cactaaracc agratatcct agttttttgg gttgactgtt ccctccccac tttccttgaa 1980 ncccaatgcc cntttgtktn ggttgcttcc ctaaaaktt 2019 <210> SEQ ID NO 25 <211> LENGTH: 350 <212> TYPE: PRT <213> ORGANISM: Mouse <220> FEATURE: <221> NAME/KEY: UNSURE <222> LOCATION: (167) <223> OTHER INFORMATION: Xaa is unsure <400> SEQUENCE: 25 Ala Arg Gly Gly Val Arg Ala Glu Ala Glu Asp Gln Val Gly Met Ala 1 5 10 15 Glu Gly Gly Thr Gly Pro Asp Gly Arg Ala Gly Pro Gly Pro Ala Gly 20 25 30 Pro Asn Leu Lys Glu Trp Leu Arg Glu Gln Phe Cys Asp His Pro Leu 35 40 45 Glu His Cys Asp Asp Thr Arg Leu His Asp Ala Ala Tyr Val Gly Asp 50 55 60 Leu Gln Thr Leu Arg Asn Leu Leu Gln Glu Glu Ser Tyr Arg Ser Arg 65 70 75 80 Ile Asn Glu Lys Ser Val Trp Cys Cys Gly Trp Leu Pro Cys Thr Pro 85 90 95 Leu Arg Ile Ala Ala Thr Ala Gly His Gly Asn Cys Val Asp Phe Leu 100 105 110 Ile Arg Lys Gly Ala Glu Val Asp Leu Val Asp Val Lys Gly Gln Thr 115 120 125 Ala Leu Tyr Val Ala Val Val Asn Gly His Leu Glu Ser Thr Glu Ile 130 135 140 Leu Leu Glu Ala Gly Ala Asp Pro Asn Gly Ser Arg His His Arg Ser 145 150 155 160 Thr Pro Val Tyr His Ala Xaa Arg Val Gly Arg Asp Asp Ile Leu Lys 165 170 175 Ala Leu Ile Arg Tyr Gly Ala Asp Val Asp Val Asn His His Leu Asn 180 185 190 Ser Asp Thr Arg Pro Pro Phe Ser Arg Arg Leu Thr Ser Leu Val Val 195 200 205 Cys Pro Leu Tyr Ile Ser Ala Ala Tyr His Asn Leu Gln Cys Phe Arg 210 215 220 Leu Leu Leu Gln Ala Gly Ala Asn Pro Asp Phe Asn Cys Asn Gly Pro 225 230 235 240 Val Asn Thr Gln Glu Phe Tyr Arg Gly Ser Pro Gly Cys Val Met Asp 245 250 255 Ala Val Leu Arg His Gly Cys Glu Ala Ala Phe Val Ser Leu Leu Val 260 265 270 Glu Phe Gly Ala Asn Leu Asn Leu Val Lys Trp Glu Ser Leu Gly Pro 275 280 285 Glu Ala Arg Gly Arg Arg Lys Met Asp Pro Glu Ala Leu Gln Val Phe 290 295 300 Lys Glu Ala Arg Ser Ile Pro Arg Thr Leu Leu Ser Leu Cys Arg Val 305 310 315 320 Ala Val Arg Arg Ala Leu Gly Lys Tyr Arg Leu His Leu Val Pro Ser 325 330 335 Leu Pro Leu Pro Asp Pro Ile Lys Lys Phe Leu Leu Tyr Glu 340 345 350 <210> SEQ ID NO 26 <211> LENGTH: 419 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 26 gcatccatgg cggagggcgg cagcacgacg ggcgggcagg gccgggctcc gcaggtcgta 60 atctgaagga gtggctgagg gagcaatttt gtgatcatcc gctggagcac tgtgaggaca 120 cgaggctcca tgatgcagct tacgtcgggg acctccagac cctcaggagc ctattgcaag 180 aggagagcta ccggagccgc atcaacgaga agtctgtctg gtgctgtggc tggctcccct 240 gcacaccgtt gcgaatcgcg gccactgcag gccatgggag ctgtgtggac ttcctcatcc 300 ggaagggggc cgaggtggat ctggtggacg taaaaggaca gacggccctg tatgtggctg 360 tggtgaacgg gcacctagag agtacccaga tccttctcga agctggcgcg gaccccaac 419 <210> SEQ ID NO 27 <211> LENGTH: 595 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 27 gaggaagaag aaaagtggac cctgaggcct tgcaggtctt taaagaggcc agaagtgttc 60 ccagaacctt gctgtgtctg tgccgtgtgg ctgtgagaag agctcttggc aaaaccggct 120 tcatctgatt ccttcgctgc ctctgccaga ccccataaag aagtttctac tccatgagta 180 gactccaagt gctgcggttg attccagtga gggagaaagt gatctgcagg gaggtggaca 240 ccgagccctg agtgctgtgc tgctgctggt ctcctgatgg ctgttgctgc agaagatgtc 300 ctcgtagact gtcattgctc ctcaggtgcc tgggccgctg aacagtcctt gggtcattgt 360 cagctgagag gcttatacta aagttattat tgtttttccc aagttctctg ttctggattt 420 tcagttgcat attaatgtaa cgggccatgg ggtatgtaca tgtaggggct gaggttggag 480 gcctactaat ttcctgtagg gaagactccc agcacttctg gaactgtgct tctctttatt 540 tttctacttc tcaatttgat ggttcgatta aagccttcta gtatctcaat gaaaa 595 <210> SEQ ID NO 28 <211> LENGTH: 896 <212> TYPE: DNA <213> ORGANISM: Mouse <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (4)..(393) <221> NAME/KEY: UNSURE <222> LOCATION: (551) <223> OTHER INFORMATION: n is unsure <221> NAME/KEY: UNSURE <222> LOCATION: (651) <223> OTHER INFORMATION: n is unsure <400> SEQUENCE: 28 ctg atg tcc gca att ctg aag gtt gga cac cac tgc tgg ctg cct gtg 48 Met Ser Ala Ile Leu Lys Val Gly His His Cys Trp Leu Pro Val 1 5 10 15 aca tcc gct gtc aat ccc caa agg atg ctg agg cca cca cca acc gct 96 Thr Ser Ala Val Asn Pro Gln Arg Met Leu Arg Pro Pro Pro Thr Ala 20 25 30 gtt ttc aac tgt gcc gct tgc tgc tgt ctg tgg ggg cag atg ctg atg 144 Val Phe Asn Cys Ala Ala Cys Cys Cys Leu Trp Gly Gln Met Leu Met 35 40 45 aat aca tac cgt gta gtt cag ctt cct gag gag gcc aag ggc ttg gtg 192 Asn Thr Tyr Arg Val Val Gln Leu Pro Glu Glu Ala Lys Gly Leu Val 50 55 60 cca cca gag att cta cag aag tac cat gga ttc tac tct tcc ctc ttt 240 Pro Pro Glu Ile Leu Gln Lys Tyr His Gly Phe Tyr Ser Ser Leu Phe 65 70 75 gcc ttg gtg agg cag ccc agg tcg ctg cag cat ctc tgc cgt tgt gcg 288 Ala Leu Val Arg Gln Pro Arg Ser Leu Gln His Leu Cys Arg Cys Ala 80 85 90 95 ctc cgc agt cac ctg gag ggc tgt ctg ccc cat gca cta ccg cgc ctt 336 Leu Arg Ser His Leu Glu Gly Cys Leu Pro His Ala Leu Pro Arg Leu 100 105 110 ccc ctg cca ccg cgc atg ctc cgc ttt ctg cag ctg gac ttt gag gat 384 Pro Leu Pro Pro Arg Met Leu Arg Phe Leu Gln Leu Asp Phe Glu Asp 115 120 125 ctg ctc tac taggcttgct gccctgtgaa caaagcagac cccaccccca 433 Leu Leu Tyr 130 ccccaagggc atctctcagc aatgaatgat gcaaggcggt ctgtcttcaa gtcaggagtg 493 gacgccttga tccacacttg agagaagagg ccagatcagc accyggctgg tagtgatngc 553 agagggcacc tgtgcagatc tgtgtgcgca ctggaaatct ctaggctgaa ggcyagagca 613 aatggtgcar gtgttagtcc ttgggangag agacagangg tgagaaagca agacagaggt 673 gagagtgcac atgtcaagtg gtagattgcc ttaaaagaaa gctaaaaaaa gaaaaagatt 733 cgggcgaact tctttagggg taatgctgca gcgtgttaaa ctgactgacc agcgtccata 793 tctttggacc cttcccgggt gaaaaagccc cttcatcctc cagcgctccc caagggtgct 853 tagcaatacc gggtgctttt ctgccgcaaa gtgagttacc aaa 896 <210> SEQ ID NO 29 <211> LENGTH: 130 <212> TYPE: PRT <213> ORGANISM: Mouse <400> SEQUENCE: 29 Met Ser Ala Ile Leu Lys Val Gly His His Cys Trp Leu Pro Val Thr 1 5 10 15 Ser Ala Val Asn Pro Gln Arg Met Leu Arg Pro Pro Pro Thr Ala Val 20 25 30 Phe Asn Cys Ala Ala Cys Cys Cys Leu Trp Gly Gln Met Leu Met Asn 35 40 45 Thr Tyr Arg Val Val Gln Leu Pro Glu Glu Ala Lys Gly Leu Val Pro 50 55 60 Pro Glu Ile Leu Gln Lys Tyr His Gly Phe Tyr Ser Ser Leu Phe Ala 65 70 75 80 Leu Val Arg Gln Pro Arg Ser Leu Gln His Leu Cys Arg Cys Ala Leu 85 90 95 Arg Ser His Leu Glu Gly Cys Leu Pro His Ala Leu Pro Arg Leu Pro 100 105 110 Leu Pro Pro Arg Met Leu Arg Phe Leu Gln Leu Asp Phe Glu Asp Leu 115 120 125 Leu Tyr 130 <210> SEQ ID NO 30 <211> LENGTH: 436 <212> TYPE: DNA <213> ORGANISM: Mouse <400> SEQUENCE: 30 gtgggggcgt catcatgacc tcctctaggg ctctgcaaca tgactcctgt ggtgcaaatc 60 aacaaattgt tcactgatga atccacaagg atctctgggc ctacaaccag gtcctggtcc 120 acatgactgt cgtcttcgga gaaggcacca ctcgcccccg gcaggtacgg ctgacacctc 180 catgggagaa gacgtatcca ggcagcagct gcgcggccct tcaagagggc acatcccgtc 240 atctaaaggc acggtgtact gaaggtagtc ctgagacatg agtccgatta ctacaggcac 300 gtgttcctcc aggtggaggc tcaggtcccc gggtgagctg gggctgcagc gggactcagg 360 gcgcggctct ggctgcaggt ctcgcagctc cctgggctgt agctcccgca gatccttgcg 420 cacaccgttg actggt 436 <210> SEQ ID NO 31 <211> LENGTH: 2180 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 31 ttaatagtac ctacatagta gaaaattata actccacttt aaaacaatgt tttctttcta 60 ttcaaatcaa tttaaaactt tttataaaca ttaatgttgc aagagaatcc agtccattta 120 tgaaaattag ttgacaatca agttcaccca agaaaatgtt gactaagcta aagaaatcac 180 agataaaaca ttttaccaaa aggataggta acacacaaaa aaatgctatc acaggaagct 240 atgatcatct aatatttctt taataataat tctagttcca taggttttca tgttatgcca 300 atttgtaccc gagtttaatt acagaaaagg caacaatttc taaattggtg gtatacattt 360 ctttacaatt ttttaatgta aggccattta ttaaaataga caaactagaa gatgaaaacg 420 aaggcaacag aaaaattcaa cttttcacaa ccaaaagaat tagcacaacc ttagaaataa 480 tttagaaaaa agtgttgtta aaagatatgt tgcagatctc cgttccatta cccaagatta 540 tgtcaattca cgattctaaa taaatctttt taaagtaaga gattaaaaac tcatcttcag 600 tgtatatgta aattccgtgg ttttatcaca caggtatgtt tattcaacac tgctttggaa 660 atggaccatt taaaaggaca tggcaatttc cattctgtta agtttcattc aacctttact 720 taggggttga ttaccacatg aaatgtgctt ttaatgcata aaaatcacag tggattagcc 780 agcaaaaggg actgggcggg gggggcattg aggagaattt gataattcac attgtgatta 840 ttctgcacat tgatgaaaca taattcacac ctctaaaacc tcaagacttc ccttttttaa 900 agaaccaaaa taaacccaag acaccttgct gacacttccc cacccctaaa caaactgatg 960 actcttttac acataaaact gaaatagtta tggcagcaaa agattttgat ggcaatgaaa 1020 gtttgtaaac tgtatttcaa tctcttgttc ttattcccaa agtgcaagat gcagggttct 1080 caatctttca gtagtgcttc tcctgtaaat aatccttcat tttgtttggc aaaggcagtt 1140 tctgaattaa gtctattctg gtatactgac gtataacaaa acgacacagg tactgcaacg 1200 agcgcaccta tgaaccccgg aacactggtt ggcaagttct gacggaagtg cagattccag 1260 gcagcgagac cttgaataac aaaaagctcc cattttcaga gtccctgatt gaatgctcca 1320 attagatcaa ctatggacgt atgtccttcc acatcggctg ttcataaaag ctaaacctac 1380 catttgagtg ctcaattcta gtgtgaagtg ttttaccatg ggagcgaaag tcacagctta 1440 aaaggtaacg gtcgtcagaa ctgtcccgaa caagaaaaga accatctggc acgtttgcta 1500 gcttcccttc tgcctcccaa cgtgtgattg gtccccagta ccatccttgc tttgcaagtt 1560 ttttcagctc ctctgtaagg cttgtcacaa ccatgggacc actactttgc actgagtcat 1620 aaactcttgc aaccccagga gcagagttcg gatcaaaatt caaatgacag cgcataactt 1680 tcagccacgt ggggctttct gtccagtgag tccactgaaa gttccccttt gggatttgga 1740 ttattcctgc attggagtaa ccaatggtga agattggagg gacatccatc gtgaacccgc 1800 tctccggggt tctgcaacat gactcccgtg gtgccaatca acaagccatt caccggactg 1860 atccacgaag atctctgggg cgacaactag gtcctggtct acctgactct catcctcggg 1920 gaaagcgcgc cctcccactt gaggaggaac cgcagagact tccatgggag aagagctgtc 1980 cagacaatag ctccgtgatc cttccaaagg atacatcccc tcatctaaag gcacagtata 2040 ctgaatgtag tcctgaggca taagtccaat aacgacaggc acatgttcat ccaggtgaag 2100 atgcaggtct ccattatgag aagccgagct cttcagtgaa ttggcttgct cctggcacgt 2160 ggtctcagac tggaggtcgt 2180 <210> SEQ ID NO 32 <211> LENGTH: 2649 <212> TYPE: DNA <213> ORGANISM: Mouse <400> SEQUENCE: 32 ggcacgaggc tgtgtccagc acacagagag ggcccggcca tctgctttgg ttcagagccc 60 tgtgtctgtc tgtcacttag actcttcctc ccggctcgca gctcaccctc catcctcctt 120 actggctcca gcatgactcg cttctcttat gcagagtact ttgctctgtt tcactctggc 180 tctgcacctt ccaggtcccc ttcgtctccc gagaacccac cggcccgcgc acccctgggt 240 ctgttccaag gggtcatgca gaagtatagc agcaacctgt tcaagacctc ccagatggcg 300 gctatggacc ccgtgctgaa ggccatcaag gaaggggatg aagaggcctt gaagatcatg 360 atccaggatg ggaagaatct tgcagagccc aacaaggagg gctggctgcc gctccacgag 420 gctgcctact atggccagct gggctgcctg aaagtcctgc agcaagccta cccagggacc 480 attgaccaac gcacactgca ggaagagaca gcattatacc tggccacatg cagagaacac 540 ctggattgcc tcctgtcgct gctccaggcg ggggcagagc ctgacatctc taacaaatcc 600 agggagactc cactttacaa agcctgtgag cgcaagaacg cggaggcggt gaggatattg 660 gtgcgataca acgcagacgc caaccaccgc tgtaacaggg gctggaccgc actgcacgag 720 tctgtctccc gcaatgacct ggaggtcatg gagatcctag tgagtggcgg ggccaaggtg 780 gaggccaaga atgtctacag catcacccct ttgtttgtgg ctgcccagag tgggcagctg 840 gaggccctga ggttcctggc caagcatggt gcagacatca acacgcaggc cagtgacagt 900 gcatcagccc tctacgaggc cagcaagaat gagcatgaag acgtggtaga gtttcttctc 960 tctcagggcg ccgatgctaa caaagccaac aaggacggcc tgctccccct gcatgttgcc 1020 tccaagaagg gcaactatag aatagtgcag atgctgctgc ctgtgaccag ccgcacgcgc 1080 gtgcgccgta gcggcatcag cccgctgcac ctagcggccg agcgcaacca cgacgcggtg 1140 ctggaggcgc tgctggccgc gcgcttcgac gtgaacgcac ctctggctcc cgagcgcgcc 1200 cgcctctacg aggaccgccg cagttctgcg ctctacttcg ctgtggtcaa caacaatgtg 1260 tacgccaccg agctgttgct gctggcgggc gcggacccca accgcgatgt catcagccct 1320 ctgctcgtgg ccatccgcca cggctgcctg cgcaccatgc agctgctgtt ggaccatggc 1380 gccaacatcg acgcctacat cgccactcac cccaccgcct ttccagccac catcatgttt 1440 gccatgaagt gcctgtcgtt actcaagttc cttatggacc tcggctgcga tggcgagccc 1500 tgcttctcct gcctgtacgg caacgggccg caccacccgc cccgcgacct ggccgcttcc 1560 acgacgcacc cgtggacgac aaggcaccta gcgtggtgca gttctgtgag ttcctgtcgg 1620 ccccggaagt gagccgctgg gcgggaccca tcatcgatgt cctcctggac tatgtgggca 1680 acgtgcagct gtgctcccgg ctgaaggagc acatcgacag ctttgaggac tgggctgtca 1740 tcaaggagaa ggcagaacct ccgagacctc tggctcacct ctgccggctg cgggttcgga 1800 aggccatagg aaaataccgg ataaaactcc tggacacact gccgcttccc ggcaggctaa 1860 tcagatactt gaaatatgag aatacacagt aaccagcctg gagaggagat gtggccttca 1920 gactgtttcc gggacgcccc aggtggcctg catccaggac cccctggggt cagaacaggt 1980 gtgaccttgc tggttctttg ctggagcttc acccaaagtg agaacctgat gtggggagtg 2040 gacgtggaac ctctgctttc acactgtcag cggatcgcag acccgctctg cttctggcca 2100 tagccagaga ccttcaacct ggggccaggg gagagctggt ctgggcaagg tggcccaggc 2160 aggaatcctg gccttaagct ggagaacttg taggaatccc tcactggacc ctcagctttc 2220 aggctgcgag ggagacgccc agcccaagta ttttatttcc gtgacacaat aacgttgtat 2280 cagaaaaaaa aaaaaacatg ggcgcagctt attccttagt agggtattta cttgcatgcg 2340 cgcttaaagc tactggaaac atgcgttcca ctatgcttga gaatcccctt gcactggtaa 2400 acgagagccg acgtgcttca aggttggatt tttggttgcc cctttggcgt tccgcgggtt 2460 tgtccgacgt aattgacccc gtgttttgtc actttcgagt gttccgacta ttggggggct 2520 tttggttgtc cccaaaattg tgggtggtgt gcggacgcca cgagaagtgg ttcatgggcg 2580 ataatcatta ctggagaatg tagagcggcg gttttacgaa taaatatttt ttaagccgcc 2640 ttcccaaaa 2649 <210> SEQ ID NO 33 <211> LENGTH: 495 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 33 cctcctgaga gttcgccggc ccgggcccaa tgggttgttc caaggggtca tgcagaaata 60 cagcagcagc ttgttcaaga cctcccagct ggcgcctgcg gaccccttga taaaggccat 120 caaggatgcg atgaagaggc cttgaagacc atgatcaagg aagggaagaa tctcgcagag 180 cccaacaagg agggctggct gccgctgcac gaggccgcat actatggcca ggtgggctgc 240 ctgaaagtcc tgcagcgagc gtacccaggg accatcgacc agcgcaccct gcaggaggaa 300 acagccgttt acttggcaac gtgcaggggc cacctggact gtctcctgtc actgctccaa 360 gcaggggcag agcgggacat ctccaacaaa tcccgagaga accgctctac aaagcctgtg 420 agcgcaagaa cgcggaagcc gtgaagattc ttggtgcagc acaacgcaga caccaacaac 480 gctgcaaccg ggctg 495 <210> SEQ ID NO 34 <211> LENGTH: 709 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 34 gtgcagctct gctcgcggct gaaggaacac atcgacagct ttgaggactg ggccgtcatc 60 aaggagaagg cagaacctcc aagacctctg gctcaccttt gccgactgcg ggttcgaaag 120 gccattggga aataccgtat aaaactccta gacaccttgc cgctcccagg caggctgatt 180 agatacctga aatacgagaa cacccagtaa ctggggccac ggggagagag gagtagcccc 240 tcagactctt cttactaagt ctcaggacgt cggtgttccc aactccaagg ggacctggtg 300 acagacgagg ctgcaggctg cctccctctc agcctggaca gctaccagga tctcactggg 360 tctcagggcc cagagctttg gccagagcag agaacagaat gtgtcaagga gaagaatcat 420 ttgtttacaa actgatgagc agatcccaga ccttctctac cttcaggaat ggcagaaacc 480 tctattcctg gggccagggc agagcttgag gtgttctggg gaaggtggtg ctcagagcct 540 tccctgtgcc cctccacttg ttctggaaaa ctcaccactt gacttcagag ctttctctcc 600 aaagactaag atgaagacgt ggcccaaggt agggggtagg gggagcctgg gtcttggagg 660 gctttgttaa gtattaatat aataaatgtt acacatgtga aaaaaaaaa 709 <210> SEQ ID NO 35 <211> LENGTH: 848 <212> TYPE: DNA <213> ORGANISM: Human <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(621) <400> SEQUENCE: 35 ttg gag aag tgt ggt tgg tat tgg ggg cca atg aat tgg gaa gat gca 48 Leu Glu Lys Cys Gly Trp Tyr Trp Gly Pro Met Asn Trp Glu Asp Ala 1 5 10 15 gag atg aag ctg aaa ggg aaa cca gat ggt tct ttc ctg gta cga gac 96 Glu Met Lys Leu Lys Gly Lys Pro Asp Gly Ser Phe Leu Val Arg Asp 20 25 30 agt tct gat cct cgt tac atc ctg agc ctc agt ttc cga tca cag ggt 144 Ser Ser Asp Pro Arg Tyr Ile Leu Ser Leu Ser Phe Arg Ser Gln Gly 35 40 45 atc acc cac cac act aga atg gag cac tac aga gga acc ttc agc ctg 192 Ile Thr His His Thr Arg Met Glu His Tyr Arg Gly Thr Phe Ser Leu 50 55 60 tgg tgt cat ccc aag ttt gag gac cgc tgt caa tct gtt gta gag ttt 240 Trp Cys His Pro Lys Phe Glu Asp Arg Cys Gln Ser Val Val Glu Phe 65 70 75 80 att aag aga gcc att atg cac tcc aag aat gga aag ttt ctc tat ttc 288 Ile Lys Arg Ala Ile Met His Ser Lys Asn Gly Lys Phe Leu Tyr Phe 85 90 95 tta aga tcc agg gtt cca gga ctg cca cca act cct gtc cag ctg ctc 336 Leu Arg Ser Arg Val Pro Gly Leu Pro Pro Thr Pro Val Gln Leu Leu 100 105 110 tat cca gtg tcc cga ttc agc aat gtc aaa tcc ctc cag cac ctt tgc 384 Tyr Pro Val Ser Arg Phe Ser Asn Val Lys Ser Leu Gln His Leu Cys 115 120 125 aga ttc cgg ata cga cag ctc gtc agg ata gat cac atc cca gat ctc 432 Arg Phe Arg Ile Arg Gln Leu Val Arg Ile Asp His Ile Pro Asp Leu 130 135 140 cca ctg cct aaa cct ctg atc tct tat atc cga aag ttc tac tac tat 480 Pro Leu Pro Lys Pro Leu Ile Ser Tyr Ile Arg Lys Phe Tyr Tyr Tyr 145 150 155 160 gat cct cag gaa gag gta tac ctg tct cta aag gaa gcg cag cgt cag 528 Asp Pro Gln Glu Glu Val Tyr Leu Ser Leu Lys Glu Ala Gln Arg Gln 165 170 175 ttt cca aac aga agc aag agg tgg aac cct cca cgt agc gag ggg ctc 576 Phe Pro Asn Arg Ser Lys Arg Trp Asn Pro Pro Arg Ser Glu Gly Leu 180 185 190 cct gct ggt cac cac caa ggg cat ttg gtt gcc aag ctc cag ctt 621 Pro Ala Gly His His Gln Gly His Leu Val Ala Lys Leu Gln Leu 195 200 205 tgaagaacca aattaagcta ccatgaaaag aagaggaaaa gtgagggaac aggaaggttg 681 ggattctctg tgcagagact ttggttcccc acgcaagccc tggggcttgg aagaagcaca 741 tgaccgtact ctgcgtgggg ctccacctca cacccacccc tgggcatctt aggactggag 801 gggctccttg gaaaactgga agaagtctca acactgtttc tttttca 848 <210> SEQ ID NO 36 <211> LENGTH: 207 <212> TYPE: PRT <213> ORGANISM: Human <400> SEQUENCE: 36 Leu Glu Lys Cys Gly Trp Tyr Trp Gly Pro Met Asn Trp Glu Asp Ala 1 5 10 15 Glu Met Lys Leu Lys Gly Lys Pro Asp Gly Ser Phe Leu Val Arg Asp 20 25 30 Ser Ser Asp Pro Arg Tyr Ile Leu Ser Leu Ser Phe Arg Ser Gln Gly 35 40 45 Ile Thr His His Thr Arg Met Glu His Tyr Arg Gly Thr Phe Ser Leu 50 55 60 Trp Cys His Pro Lys Phe Glu Asp Arg Cys Gln Ser Val Val Glu Phe 65 70 75 80 Ile Lys Arg Ala Ile Met His Ser Lys Asn Gly Lys Phe Leu Tyr Phe 85 90 95 Leu Arg Ser Arg Val Pro Gly Leu Pro Pro Thr Pro Val Gln Leu Leu 100 105 110 Tyr Pro Val Ser Arg Phe Ser Asn Val Lys Ser Leu Gln His Leu Cys 115 120 125 Arg Phe Arg Ile Arg Gln Leu Val Arg Ile Asp His Ile Pro Asp Leu 130 135 140 Pro Leu Pro Lys Pro Leu Ile Ser Tyr Ile Arg Lys Phe Tyr Tyr Tyr 145 150 155 160 Asp Pro Gln Glu Glu Val Tyr Leu Ser Leu Lys Glu Ala Gln Arg Gln 165 170 175 Phe Pro Asn Arg Ser Lys Arg Trp Asn Pro Pro Arg Ser Glu Gly Leu 180 185 190 Pro Ala Gly His His Gln Gly His Leu Val Ala Lys Leu Gln Leu 195 200 205 <210> SEQ ID NO 37 <211> LENGTH: 464 <212> TYPE: DNA <213> ORGANISM: Mouse <400> SEQUENCE: 37 gttccaagcc taacccatct ttgtcgtttg gaaattcggg ccagtctaaa agcagagcac 60 cttcactctg acattttcat ccatcagttg ccacttccca gaagtctgca gaactatttg 120 ctctatgaag aggttttaag aatgaatgag attctagaac cagcagctaa tcaggatgga 180 gaaaccagca aggccacctg acacaggtcc tttaattctg tttagtcaca aaagacggct 240 tgtgtgactg tttggatttg gtgatcaaat gtccatgttt acagttgctt ttcccagttt 300 gtgtctttcc caatattgtg aaccttatcc atcttgcctt actcagtttt atttctagtg 360 cactttgttg tgtattattt gtttacctga ccattttcta ctttattctg ctaataaact 420 gtaattctga aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 464 <210> SEQ ID NO 38 <211> LENGTH: 747 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 38 ggggatcgaa agcgggggct tctgggacgc agctctggag acgcggcctc ggaccagcca 60 tttcggtgta gaagtggcag cacggcagac tggtcaaaca aatggatttt acagaggctt 120 acgcggacac gtgctctaca gttggacttg ctgccaggga aggcaatgtt aaagtcttaa 180 ggaaactgct caaaaagggc cgaagtgtcg atgttgctga taacagggga tggatgccaa 240 ttcatgaagc agcttatcac aactctgtag aatgtttgca aatgttaatt aatgcagatt 300 catctgaaaa ctacattaag atgaagacct ttgaaggttt ctgtgctttg catctcgctg 360 caagtcaagg acattggaaa atcgtacaga ttcttttaga agctggggca gatcctaatg 420 caactacttt agaagaaacg acaccattgt ttttagctgt tgaaaatgga cagatagatg 480 tgttaaggct gttgcttcaa cacggagcaa atgttaatgg atcccattct atgtgtggat 540 ggaactcctt gcaccaggct tcttttcagg aaaatgctga gatcataaaa ttgcttctta 600 gaaaaggagc aaacaaggaa tgccaggatg actttggaat cacaccttta tttgtggctg 660 ctcagtatgg ccaagctaga aagctttgaa gcatacttat ttcatccggg tgcaaatgtc 720 aattgtcaag ccttggacaa agctacc 747 <210> SEQ ID NO 39 <211> LENGTH: 1018 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 39 cacaaatggg accatacaaa aatcttggac ttgttaataa ccacttacta accgggacct 60 gtgacactgg gctaaacaaa gtaagtccct gtttactcag cagtgtttgg gggacatgaa 120 ggattgccta gaaatattac tccggaatgg tctacagccc agacgcccag gcgtgccttg 180 tttttggatt cagttctcct gtgtgcatgg ctttccaaaa ggaggtggag ctgtagttct 240 ttggaattgt gaacattctt ttgaaatatg gagcccagat aaatgaactt catttggcat 300 actgcctgaa gtacgagaag ttttcgatat ttcgctactt tttgaggaaa ggttgctcat 360 tgggaccatg gaaccatata tatgaatttg taaatcatgc aattaaagca caagcaaaat 420 ataaggagtg gttgccacat cttctggttg ctggatttga cccactgatt ctactgtgca 480 attcttggat tgactcagtc agcattgaca cccttatctt cactttggag tttactaatt 540 ggaagacact tgcaccagct gttgaaagga tgctctctgc tcgtgcctca aacgcttgga 600 ttctacagca acatattgcc cactgttcca tccctgaccc atctttgtcg tttggaaatt 660 cggtccagtc taaaatcaga acgtctacgg tctgacagtt atattagtca gctgccactt 720 cccagaagcc tacataatta tttgctctat gaagacgttc tgaggatgta tgaagttcca 780 gaactggcag ctattcaaga tggataaatc agtgaaacta cttaacacag ctaatttttt 840 tctctgaaaa atcatcgaga caaaagagcc acagagtaca agtttttatg attttatagt 900 caaaagatga ttattgattg tcagataggt taggttttgg ggggccagta gttcagtgag 960 aatgtttatg tttacaacta gccttcccag taaaaaaaaa aaaaaaaaaa aaaaaaaa 1018 <210> SEQ ID NO 40 <211> LENGTH: 1897 <212> TYPE: DNA <213> ORGANISM: Mouse <400> SEQUENCE: 40 cggggggctg ggacctgggg cgtaaccgtc tctaccacga cggcaagaac cagccaagta 60 aaacataccc agcctttctg gagccggacg agacattcat tgtccctgac tcctttttcg 120 tggccctgga catgratgat gggaccttaa gtttcatcgt ggatggacag tacatgggag 180 tggctttccg gggactcaag ggtaaaaagc tgtatcctgt agtgagtgcc gtctggggcc 240 actgtgagat ccgcatgcgc tacttgaacg gacttgatcc tgagcccctg ccactcatgg 300 acctgtgccg gcgttcggtg cgcctagcgc tgggaaaaga gcgcctgggt gccatccccg 360 ctctgccgct acctgcctcc ctcaaagcct acctcctcta ccagtgatcc acatcccagg 420 accgccatac gacagccatc tggtgccaar tcactgagcc cgttggggtc cgccgacccc 480 tgcgcctggg atggaagccc acctcagcca tgggcagacg tgccccctca tcctaccggc 540 tgcctctgct gggggaacct atgccaacgg acttctccct tcccaacact ggctgaagca 600 gcagcaccca ggcccttccc tgaaccagat gcagagaata aactatgaaa acctctctca 660 ggcgccttct gctctcaggt ggagtgggct gccccccact ctctgcagag agaggctaca 720 cccacctggg gggtcctggg aggtaagact agtaggaggt gccagggctg artccaaaag 780 caggaatggc caggamcagg ccatacagat gaagctcagg atgtcacata ccatggacam 840 tgagacagaa ccccaggttg gamttccctt gggccaacga gtgccagctt taatgtcagc 900 tgcmggtgct ctgtggcctg tatttattct ttaaacagta gcaaaggcca tttatttatt 960 ccacttagaa aggaaacctt ggtgggtggy ttccctcgat gtgctttccc ccacctccct 1020 ggaatgtgtg tgccacacct gtccttgtcc caggccagga ctgtggcaca tgagctggtg 1080 tgcacagata cacgtatgtc gtcgtgcatg acccctgact agttcctaag tagccctgca 1140 ccaagcacca gagcagaccc caagagaggc ccgtgcaagt ccccatgtcc ccaggtccct 1200 gcttctgttg ccttgggact catacaccgg cacacgtgtt tcagcctctt gacttccatg 1260 agcttcgaat tttgcccccg attcttctga tatttcccat tggcatcctc caaagctctg 1320 ggcctggagg gcattaggac acatggaatg agtggggtct ccagcccctg ggaaagccac 1380 tggcaaggca ggattagaaa gaccaagagc agggtggggc gccatgaagc ctgtatgcct 1440 ctcaggctca agaccccgcc acacacccac tcaagcctca gaagtggtgt gtagggcagc 1500 cccaggagag gaatgcctgt cctagcagca cgtacatgga gcaccccaca tgtgctccag 1560 ccctctggct gtttctcttg ctctagaatc aactccctac attgggaatg tagccatttg 1620 gtagaggact tgcctagcct gcaggaagct cacgttccat cccctgcacc aaggagaatc 1680 aaagctcagg aggctgaggc aggaggattg ctgtcagtgg tgtacagagg tcatggccat 1740 cctgggctat attaaacctt gtcctttaag aaaaagaaaa gaaatcaact tccattgaat 1800 ctgagttctg ctcatttctg cacaggtaca atagatgact tkatttgttg aaaaatgktt 1860 aatatattta cmtatatata tatttgtaag aagcatt 1897 <210> SEQ ID NO 41 <211> LENGTH: 134 <212> TYPE: PRT <213> ORGANISM: Mouse <220> FEATURE: <221> NAME/KEY: UNSURE <222> LOCATION: (45) <223> OTHER INFORMATION: Xaa is unsure <400> SEQUENCE: 41 Gly Gly Trp Asp Leu Gly Arg Asn Arg Leu Tyr His Asp Gly Lys Asn 1 5 10 15 Gln Pro Ser Lys Thr Tyr Pro Ala Phe Leu Glu Pro Asp Glu Thr Phe 20 25 30 Ile Val Pro Asp Ser Phe Phe Val Ala Leu Asp Met Xaa Asp Gly Thr 35 40 45 Leu Ser Phe Ile Val Asp Gly Gln Tyr Met Gly Val Ala Phe Arg Gly 50 55 60 Leu Lys Gly Lys Lys Leu Tyr Pro Val Val Ser Ala Val Trp Gly His 65 70 75 80 Cys Glu Ile Arg Met Arg Tyr Leu Asn Gly Leu Asp Pro Glu Pro Leu 85 90 95 Pro Leu Met Asp Leu Cys Arg Arg Ser Val Arg Leu Ala Leu Gly Lys 100 105 110 Glu Arg Leu Gly Ala Ile Pro Ala Leu Pro Leu Pro Ala Ser Leu Lys 115 120 125 Ala Tyr Leu Leu Tyr Gln 130 <210> SEQ ID NO 42 <211> LENGTH: 265 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 42 aagggtaaaa aactgtatcc tgtagtgagt gccgtctggg gccactgtag atccgaatgc 60 gctacttgaa cggactcgat cccgagactg ccgctcatgg atttgtgccg tcgctcggtg 120 cgcctggccc tggggaggga gcgcctgggg gagaaccaca cctgccgctg ccggcttccc 180 tcaaggccta cctcctctac cagtgacgtt cgccatcata ccgccagcgc gacagccacc 240 tggtgccaac tcactgagcc gcctg 265 <210> SEQ ID NO 43 <211> LENGTH: 2438 <212> TYPE: DNA <213> ORGANISM: Mouse <400> SEQUENCE: 43 aagtggcggc ggtccctgga gagcaggcgg aggcagcggc aagtctgact ctgggctgac 60 cgtggagccg gggcgggggc tgacagccag gcctccgcct ggcgggagcc gcacgaggag 120 cgggagtggc cgggcctctc ttccgcgctt gagcgagcgc cgggtgatgg cggtggtgat 180 ggcggcaggc gctcggacag ctccgcttga gctgagctcg gagagatccg tccagaaagt 240 gcccagaaga aacttcctct tagaaaagct gaaaaacaca rtatttataa cactggaaat 300 tgtaaagaat ttgtttaaaa tggctgaaaa caatagtaaa aatgtagatg tacggcctaa 360 aacaagtcgg agtcgaagtg ctgacaggaa ggatggttat gtgtggagtg gaaagaagtt 420 gtcttggtcc aaaaagagtg agagttgttc tgaatctgaa gccataggta ctgttgagaa 480 tgttgaaatt cctctaagaa gccaagaaag gcagcttagc tgttcgtcca ttgagttgga 540 cttagatcat tcctgtgggc atagattttt aggccgatcc cttaaacaga aactgcaaga 600 tgcggtgggg cagtgttttc caataaagaa ttgtagtggc cgacactctc cagggcttcc 660 atctaaaaga aagattcata tcagtgaact catgttagat aagtgccctt tcccacctcg 720 ctcagattta gcctttaggt ggcattttat taaacgacac actgttccta tgagtcccaa 780 ctcagatgaa tgggtgagtg cagacctgtc tgagaggaaa ctgagagatg ctcagctgaa 840 acgaagaaac acagaagatg acataccctg tttctcacat accaatggcc agccttgtgt 900 cataactgcc aacagtgctt cgtgtacagg tggtcacata actggttcta tgatgaactt 960 ggtcacaaac aacagcatag aagacagtga catggattca gaggatgaaa ttataacgct 1020 gtgcacaagc tccagaaaaa ggaataagcc caggtgggaa atggaagagg agatcctgca 1080 gttggaggca cctcctaagt tccacaccca gatcgactac gtccactgcc ttgttccaga 1140 cctccttcag atcagtaaca atccgtgcta ctggggtgtc atggacaaat atgcagccga 1200 agctctgctg gaaggaaagc cagagggcac ctttttactt cgagattcag cgcaggaaga 1260 ttatttattc tctgttagtt ttagacgcta cagtcgttct cttcatgcta gaattgagca 1320 gtggaatcat aactttagct ttgatgccca tgatccttgt gtcttccatt ctcctgatat 1380 tactgggctc ctggaacact ataaggaccc cagtgcctgt atgttctttg agccgctctt 1440 gtccactccc ttaatccgga cgttcccctt ttccttgcag catatttgca gaacggttat 1500 ttgtaattgt acgacttacg atggcatcga tgcccttccc attccttcgc ctatgaaatt 1560 gtatctgaag gaataccatt ataaatcaaa agttaggtta ctcaggattg atgtgccaga 1620 gcagcagtga tgcggagagg ttagaatgtc gacctgcata catattttca tttaatattt 1680 tatttttctt atgcctcttt gaatttttgt acaaaggcag ttgaatcaaa taaaactgtg 1740 ccctaagttt taattccaga tcaatttatt ttttttatga tacacttgtt atatattttt 1800 aagcaggtgt ttggttttgt ttttaccata taaatttaca tatggtccag gcatatttac 1860 aatttcaagg cattgcatat acatttgaat attctgtatt ttttaaataa tcttttgttc 1920 tttcctatgt gtgaaatatt ttgctaatct atgctatcag tattcttgta tgaccgaata 1980 gttacctatt ctcttttcat cttgaagatt ttcagtaaag agtgttgtaa tcaatccatt 2040 ataatgtaat tgacttttgt aatttgccaa taggagtgtt aaacaacaaa atgatttaaa 2100 atgaaactta atgtattttc attttaaata ttaactaaac caagtttgtt tgttagttat 2160 tctagccaat aagaaaagag aatgtagcat cctagaggtg tatttgttct gcagtttggc 2220 aggaccgtca gttagtccaa ataaacatcc cctcagcgtg gaggcgaatg gaacctgtgc 2280 tcctttctta cgggaagctt tgcaaagcaa aatagcaggg ttacaagctt ggagttgtta 2340 aggcaactag agttttctct attaatttat agactgttgt tgcacctact tagctctttt 2400 ttgggaactc tagttcccag gggaaaatac ctcgtgcc 2438 <210> SEQ ID NO 44 <211> LENGTH: 542 <212> TYPE: PRT <213> ORGANISM: Mouse <220> FEATURE: <221> NAME/KEY: UNSURE <222> LOCATION: (94) <223> OTHER INFORMATION: Xaa is unsure <400> SEQUENCE: 44 Ser Gly Gly Gly Pro Trp Arg Ala Gly Gly Gly Ser Gly Lys Ser Asp 1 5 10 15 Ser Gly Leu Thr Val Glu Pro Gly Arg Gly Leu Thr Ala Arg Pro Pro 20 25 30 Pro Gly Gly Ser Arg Thr Arg Ser Gly Ser Gly Arg Ala Ser Leu Pro 35 40 45 Arg Leu Ser Glu Arg Arg Val Met Ala Val Val Met Ala Ala Gly Ala 50 55 60 Arg Thr Ala Pro Leu Glu Leu Ser Ser Glu Arg Ser Val Gln Lys Val 65 70 75 80 Pro Arg Arg Asn Phe Leu Leu Glu Lys Leu Lys Asn Thr Xaa Phe Ile 85 90 95 Thr Leu Glu Ile Val Lys Asn Leu Phe Lys Met Ala Glu Asn Asn Ser 100 105 110 Lys Asn Val Asp Val Arg Pro Lys Thr Ser Arg Ser Arg Ser Ala Asp 115 120 125 Arg Lys Asp Gly Tyr Val Trp Ser Gly Lys Lys Leu Ser Trp Ser Lys 130 135 140 Lys Ser Glu Ser Cys Ser Glu Ser Glu Ala Ile Gly Thr Val Glu Asn 145 150 155 160 Val Glu Ile Pro Leu Arg Ser Gln Glu Arg Gln Leu Ser Cys Ser Ser 165 170 175 Ile Glu Leu Asp Leu Asp His Ser Cys Gly His Arg Phe Leu Gly Arg 180 185 190 Ser Leu Lys Gln Lys Leu Gln Asp Ala Val Gly Gln Cys Phe Pro Ile 195 200 205 Lys Asn Cys Ser Gly Arg His Ser Pro Gly Leu Pro Ser Lys Arg Lys 210 215 220 Ile His Ile Ser Glu Leu Met Leu Asp Lys Cys Pro Phe Pro Pro Arg 225 230 235 240 Ser Asp Leu Ala Phe Arg Trp His Phe Ile Lys Arg His Thr Val Pro 245 250 255 Met Ser Pro Asn Ser Asp Glu Trp Val Ser Ala Asp Leu Ser Glu Arg 260 265 270 Lys Leu Arg Asp Ala Gln Leu Lys Arg Arg Asn Thr Glu Asp Asp Ile 275 280 285 Pro Cys Phe Ser His Thr Asn Gly Gln Pro Cys Val Ile Thr Ala Asn 290 295 300 Ser Ala Ser Cys Thr Gly Gly His Ile Thr Gly Ser Met Met Asn Leu 305 310 315 320 Val Thr Asn Asn Ser Ile Glu Asp Ser Asp Met Asp Ser Glu Asp Glu 325 330 335 Ile Ile Thr Leu Cys Thr Ser Ser Arg Lys Arg Asn Lys Pro Arg Trp 340 345 350 Glu Met Glu Glu Glu Ile Leu Gln Leu Glu Ala Pro Pro Lys Phe His 355 360 365 Thr Gln Ile Asp Tyr Val His Cys Leu Val Pro Asp Leu Leu Gln Ile 370 375 380 Ser Asn Asn Pro Cys Tyr Trp Gly Val Met Asp Lys Tyr Ala Ala Glu 385 390 395 400 Ala Leu Leu Glu Gly Lys Pro Glu Gly Thr Phe Leu Leu Arg Asp Ser 405 410 415 Ala Gln Glu Asp Tyr Leu Phe Ser Val Ser Phe Arg Arg Tyr Ser Arg 420 425 430 Ser Leu His Ala Arg Ile Glu Gln Trp Asn His Asn Phe Ser Phe Asp 435 440 445 Ala His Asp Pro Cys Val Phe His Ser Pro Asp Ile Thr Gly Leu Leu 450 455 460 Glu His Tyr Lys Asp Pro Ser Ala Cys Met Phe Phe Glu Pro Leu Leu 465 470 475 480 Ser Thr Pro Leu Ile Arg Thr Phe Pro Phe Ser Leu Gln His Ile Cys 485 490 495 Arg Thr Val Ile Cys Asn Cys Thr Thr Tyr Asp Gly Ile Asp Ala Leu 500 505 510 Pro Ile Pro Ser Pro Met Lys Leu Tyr Leu Lys Glu Tyr His Tyr Lys 515 520 525 Ser Lys Val Arg Leu Leu Arg Ile Asp Val Pro Glu Gln Gln 530 535 540 <210> SEQ ID NO 45 <211> LENGTH: 5000 <212> TYPE: DNA <213> ORGANISM: Mouse <400> SEQUENCE: 45 ccctctgggc aagccgcccc ccccccaccc atctaccaca cacacacaca cacacacaca 60 cacacattca gaccttgggg caaaaacaaa gcaaaataac aacaacaaaa acactgcctg 120 tggaaagtcc ttacttcagg aaggttggca gatgaggagc aagggaacat tttatcagga 180 ctgccacaaa ggagtctttt tttttaatgg tttttcaaga cagggtttct ctgtatagcc 240 ctggctgtcc tggagctcac tttgtagacc aggctggcct cgaactcaga aattcgcctg 300 cctctgcctc ctgagtgctg ggattaaagg cgtgcagcac catgtccaac tggcattttc 360 tcaattaagg ttcgttcctt tcagataact ctaggttctg ggtcaagctg acacaaggct 420 acacagcaca gtttgtatgc cacattcagt tcagaagaca cccaacctcc ctggaactgg 480 aacttatgca catttgtgag cttccacttg ggagtgggaa cctgaactgg gtcctctgca 540 agagcagccg tgctcttaac tgctgagcca tttcagcagc ctcacatcag aattaagtta 600 gaaattagcc gggtatgaat cataccctta gaatcctagc atctgaaagc agagctaaga 660 gaaacaggga ttcaagacca gctcttggct acagagcccg tcctgtccta ggatgggcta 720 caagagacta tttcaaagcc atccaaacaa caataactac aacaacaaca aggttaaaat 780 taggctgggc acagggtaca cacctttaat gccaacactc aggaggcaga ggcaggctga 840 tcagtgtgag tttgagttca acgtggtcta catagggagt tctaggccag cagaggttac 900 agtctctctc tctctctctc tctctctctc tctctcacac acacacacac acacacacac 960 acacacacac acacacacgg tggcattatg ggattttttt gggataaggt ttctctgtct 1020 agccctggca tagattcact ctgtagacta ggctagcctt gaactcagag atccgcctgc 1080 ctctgcctcc caagtgctgg gattataggt gttgcaccac cactgcccag ccactttggg 1140 atttttgaac tgttatcaag aggctttcga ggaggtcaaa cttcaacagc aacctctcca 1200 tgataatgta gctaatgatc aaacgacact caaaacttaa cccttaaagc acacatccac 1260 cagacagcgt gcccactcgt agttccatta ctcaggaggc tgaagcagga ggatgaagga 1320 ctaaggcttc agcaacctag ggagccgcag gggacagtag tctcaatccc tacattctcc 1380 tgaacacagg agcaggagtt caggaagggt gtcaaggccg cttactgatc ttagggcctc 1440 aggaatgact agctcaggca gagagagcaa aggtctccag tggagaagtc tacacacaca 1500 cacacacaca cacacacaca cacacacaca cagaatccaa ggcgatgacg tcatcaaagg 1560 gttaattcta gtctgggatg ggggggaggg tggggcacgc agctgtcagg tggctttgga 1620 aaaataaact gctgaagagt ctgacgccag ggagtcctgg gagggacaag aggttaccca 1680 ctcaaagagt gtgctccaca aagcatgcgc gcttgtccac gtctggagtc gtcacttatt 1740 ttttgcctgg attctttgta gccggtgggt tctcaaggcg gtaagtggtg tggccgccgt 1800 ggtctgggag gtgacgatag ggttaatcgt ccacagagcc caggggcgga gcgcgggcgg 1860 gcgtccgcag ccccgctgga gccggaagca gtggctggtc aggggcgctt ctagccttcc 1920 ctatctgtac ttccacagag gtctctgcga gctaggggga cagtgaggtg cggggtaggg 1980 gcccggcgtt agagccagca aggggacggt tcacggtaag gtctgaggga gagagagctc 2040 ctgagaaact tggggggcgc gacacagata gggtgaaagc agagtgatag acctgggatg 2100 gttaggggac caagggaaga ccaggctggt tggcatacac cggtgaacgg atgggagtcc 2160 tagggaaaga tgatgcgcct aacagtcctt tctgtctcca caccactcca ggggacgatc 2220 cggagctcaa ctttcaaaag cgagacgccc cagcaagcct gttttgagaa gttcttcagc 2280 ggctctcctc atgggccaga cggccctggc aaggggcagc agcagcaccc ctacctcgca 2340 ggctctgtac tcggacttct ctcctcccga gggcttggag gagctcctgt ctgctccccc 2400 tcctgacctg gttgcccaac ggcaccacgg ctggaacccc aaggattgct ccgagaacat 2460 cgatgtcaag gaagggggtc tgtgctttga gcggcgccct gtggcccaga gcactgatgg 2520 agtccggggg aaacggggct attcgagagg tctgcacgcc tgggagatca gctggcccct 2580 ggagcaaagg ggcacacacg ccgtggtggg cgtggccacc gccctcgccc cgctgcaggc 2640 tgaccactat gcggcgcttt tgggcagcaa cagcgagtcc tggggctggg atattgggcg 2700 gggaaaattg tatcatcaga gtaagggcct cgaggccccc cagtatccag ctggacctca 2760 gggtgagcag ctagtggtgc cagagagact gctggtggtt ctggacatgg aggaggggac 2820 tcttggctac tctattgggg gcacgtacct gggaccagcc ttccgtggac tgaaggggag 2880 gaccctctat ccctctgtaa gtgctgtttg gggccagtgc caggtccgca tccgctacat 2940 gggcgaaaga agaggtgaga tacggactag gtgtggggag atcactactc ttggcaatgg 3000 tttgggctgg aaactcatgg ttggagcaca ggaagtaggc ttcttgtcac tttggcctgt 3060 cacttagatg gccttggatc tagcttcact cccaatccct attggatgtg atgcacaaat 3120 tcagagcctt tgggtctccc tcagctgagg tggcggtgga aatggaggaa gaaggaaggg 3180 tgcctgagca ggatctcaag ttcaaggatg cctggagttg cttacttacc ttgtcttcct 3240 tctctctccg cagtggagga accacaatcc cttctgcacc tgagccgcct gtgtgtgcgc 3300 catgctctgg gggacacccg gctgggtcaa atatccactc tgcctttgcc ccctgccatg 3360 aagcgctatc tgctctacaa atgacccagt agtacagggt gtgctggcac cctaccgtgg 3420 ggacaggtgg agaggcaccc gctggcctag acaactttaa aaagctggtg aagctggggg 3480 gggggggctg gaccccttca cctccccttc tcacaggagc aagacatata gaaatgatat 3540 taaacaccat ggcagcctgg gacaaagagg tttttgaagt aaaaaatgag atgtattgtc 3600 acaacctgtt tcattattgt tttttgtttt gttttacact cccccacccc aggctagagc 3660 cccatcactg tcttaaggaa ttatgacaac ccacaaagct caggcccagg tgtttatttc 3720 ccttacatgt aggatggttc acaaacacaa tacaggggct ttggcaccgt gggggagggg 3780 actatcccag gcctcttagg gtctcatgta taccgaattc agacccgaaa gctctgaatt 3840 tctgcatcag acatccagta gaacttggga gtgaagctag agccaaggcc atctaagtga 3900 caggccaaag tgacacgaag cccacttcct gtgctccaac catgagtttc cagcccaaac 3960 caatggaagg tgatttcact tgtcagggcc caaagggaca gtcagttcta ctccctcccc 4020 tcactaggag ccaccttggt gacagttgat tctacccact gtaagtggta aagggattgg 4080 cctggtccca accataatag ggcggtggaa acggctcagg agggtacagc gtggattagg 4140 ccacaagatg gggcagatga tgtcatcaga agcatgtgac cggtgggagc agttactaaa 4200 cttctgggca acctagtcca tgctatgcag gcaggtagag ggatgggcag tgctcattgt 4260 ttggcattga tgatgtccac aaattcaggc ttgagagatg cgccacccac aaggaagccg 4320 tccacgtcag gctggcttgc cagctctttg caggttgctc cagtcacaga acctgtacca 4380 ggaacaagaa gacagtttgg tcaggtctat gatcagaaca cttaagcccc acctctctgt 4440 gcaaggcagc ctcagtctgt cttagcccat ttccgtctta gctagagcca aagccactca 4500 cctccataaa tgatccgggt gctctgagcc accccatcat tgacattgga tttcagccat 4560 ccccggagct tctcgtgtac ttcctgtgcc tagaaggagg aggcagagct actaagtaag 4620 ctccttccta tctatcattc aaggagtaaa aaccactggt tctcacatag agttgagttt 4680 ccagaaaagc cccgggacca gagagtggca aggctccaat cccaccaggc ttggaatgaa 4740 catttttggc aaagtcactc tccttggtga gtttgggggc cctctgtctc taaaggggct 4800 tggatgggct ccatagctgt gtgagtctgt taaagccgga caggctgagg agctctgggt 4860 agttacctgc tgaggggttg ccgtcttgcc agtcccaatg gcccacacag gttcataggc 4920 caggaccacc ttgctccagt ctttcacatt atctgtgggg cagagaggag agtgagtagg 4980 aaggagctga cccgccaagc 5000 <210> SEQ ID NO 46 <211> LENGTH: 264 <212> TYPE: PRT <213> ORGANISM: Mouse <400> SEQUENCE: 46 Met Gly Gln Thr Ala Leu Ala Arg Gly Ser Ser Ser Thr Pro Thr Ser 1 5 10 15 Gln Ala Leu Tyr Ser Asp Phe Ser Pro Pro Glu Gly Leu Glu Glu Leu 20 25 30 Leu Ser Ala Pro Pro Pro Asp Leu Val Ala Gln Arg His His Gly Trp 35 40 45 Asn Pro Lys Asp Cys Ser Glu Asn Ile Asp Val Lys Glu Gly Gly Leu 50 55 60 Cys Phe Glu Arg Arg Pro Val Ala Gln Ser Thr Asp Gly Val Arg Gly 65 70 75 80 Lys Arg Gly Tyr Ser Arg Gly Leu His Ala Trp Glu Ile Ser Trp Pro 85 90 95 Leu Glu Gln Arg Gly Thr His Ala Val Val Gly Val Ala Thr Ala Leu 100 105 110 Ala Pro Leu Gln Ala Asp His Tyr Ala Ala Leu Leu Gly Ser Asn Ser 115 120 125 Glu Ser Trp Gly Trp Asp Ile Gly Arg Gly Lys Leu Tyr His Gln Ser 130 135 140 Lys Gly Leu Glu Ala Pro Gln Tyr Pro Ala Gly Pro Gln Gly Glu Gln 145 150 155 160 Leu Val Val Pro Glu Arg Leu Leu Val Val Leu Asp Met Glu Glu Gly 165 170 175 Thr Leu Gly Tyr Ser Ile Gly Gly Thr Tyr Leu Gly Pro Ala Phe Arg 180 185 190 Gly Leu Lys Gly Arg Thr Leu Tyr Pro Ser Val Ser Ala Val Trp Gly 195 200 205 Gln Cys Gln Val Arg Ile Arg Tyr Met Gly Glu Arg Arg Val Glu Glu 210 215 220 Pro Gln Ser Leu Leu His Leu Ser Arg Leu Cys Val Arg His Ala Leu 225 230 235 240 Gly Asp Thr Arg Leu Gly Gln Ile Ser Thr Leu Pro Leu Pro Pro Ala 245 250 255 Met Lys Arg Tyr Leu Leu Tyr Lys 260 <210> SEQ ID NO 47 <211> LENGTH: 5615 <212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 47 gtactttctt tatatctcca taattttatt tactattact acatgataca ttattttata 60 aaagtctttg taacctcctt aaggattcac tgcttaatct ccagtgctta gcacaaatca 120 ttaaatgcga accagaaact cttccaaatg tgttacatct ataacctcat tggattctca 180 ctaccaaccc catgcaatag atactaatgt gatctctgtc ttacagagga agaaacaggc 240 acagggaggt tcagtaattt gcccaaggtc atacacacac tggccttcag gtattcatgc 300 ccggggagtc tggtcccaca gctggcatgt ttgccattat attatattgc ctccttatag 360 tgtcggcact cattaagcac attgacagct atgcttggtg agtgactact atgtacccag 420 ctctgtgcta catgctttac ctggattatt tcaactgcac aacaaccctg tgaggtaact 480 accatcattg ctcctatttt acataacaga aaactacaga aatctggggc tgggcgtagt 540 ggctcatgcc tgaaatccca gcactttggg agaccctgtc tctaaaaaaa attttttttt 600 ggccggacgt ggtggctcac acctgtaatc tcagcacttt gggaggctaa ggcaggcaga 660 tcacaaggtc aggagttcta gaccagcctg gccaacatgg caaaaccctg tgtctactaa 720 aaatacaaaa aatagctagg cgtggtggca ggtgcctgta atcccagcta ctcaggaggc 780 tgaggcagga gaatcccctg aacctgggag atggaggtta cagagagccg agatcgtgcc 840 gctgcactcc agcctgggca acaagagcaa gactctgtct cgaaaaaaat aaaaataaaa 900 ataaaaatat ttttttaaaa attagctggg tgtggtagca catgcctgta gtcccagcta 960 cttgggaggc tgaggtagga ggatcacttg agcccaggag gtcaaggctg cagtgggctg 1020 tgatggcgcc actgcactct agccttggtg acagcaagac cctgtctcaa aaaaaaaaaa 1080 aagagaaatc gggcaacttc cccaagatcg cgcagttaac tagtggcata gcttcactca 1140 aactcgaagt cttaatcagg acactctacc aaatgagatc aacggctcag taatggattg 1200 gcatccagta tgaagactgg accagcaggg agaactatga tgcgtacagc ctagagcctg 1260 aagcagattt cacagcctca gaggtggcac aggctgactc acaacccggg gcagaaaggg 1320 accagcccag aaacagtgac ccagaatcac agggaagtag aaatgggatt cggcacaatg 1380 aagcccctcc ttgaccccat gctccttacc ctcaggggcg caggagttag tcgctcaggc 1440 ggctcaaagg tcttgacggt ggagaacacc atccccaggg attcccgacg cggtgatgcc 1500 atcaaagcgt taattctgag atgggcctgc ccgggtgcgg actctgccgc agcaagagaa 1560 gggttaactg ccccgggcct tcgccgtggg ggcggggcct cggggagggt cacagcccgg 1620 gactgagacc cgaggttaac cgcccggggt gggctccacg ggggcggggc atgctctccg 1680 cggctgctgc cggtatagag cggtaactgc ccaggagggg gcggggcccc acaggggcgt 1740 ggcctcggag ctgcacggcc gtgggcggcg atgagagggt taagccccag agggccctgg 1800 aggggcgggg ccgcgggacg ggctcggccc aagggaggag ctgggggcgg aagcggccgg 1860 cggtctgcgc cctgcgcgcc tcggcttctt tccgcccggc tccttcagag gcccggcgac 1920 ctccagggct gggaagtcaa ccgaggttcg ggggcagcgg cgagggctcc gggcgagtaa 1980 gggggatggt ccatgctgag gcccaaatgg ggcgaactcg cgagagtctc tggcgacctg 2040 gatcagatgg ggcgagggca gatgaagggc ccaggagctt tggggcagcg aggagggagg 2100 agcgggcccg ttggcaaact tgggtgaaag gatggggtac ctgggtgacg agcccccgcc 2160 aggattctgc tcttcacgcc ccttttctcc cagctccctt ccaggtcaat ccaaactgga 2220 gctcaacttt cagaagagaa agacgcccca gcaagcctct ttcggggagt cctctagctc 2280 ctcacctcca tgggccagac agctctggca gggggcagca gcagcacccc cacgccacag 2340 gccctgtacc ctgacctctc ctgtcccgag ggcttggaag agctgctgtc tgcaccccct 2400 cctgacctgg gggcccagcg gcgccacggt tggaacccca aagactgttc agagaacatc 2460 gaggtcaagg aaggagggtt gtactttgag cggcggcccg tggcccagag cactgatggg 2520 gcccggggta agaggggcta ttcaaggggc ctgcacgcct gggagatcag ctggccccta 2580 gagcagaggg gcacgcatgc cgtggtgggc gtggccacgg ccctcgcccc gctgcagact 2640 gaccactacg cggcgctgct gggcagcaac agcgagtcgt ggggctggga catcgggcgg 2700 gggaagctgt accatcagag caaggggccc ggagcccccc agtatccagc gggaactcag 2760 ggtgagcagc tggaggtgcc agagagactg ctggtggttc tggacatgga ggagggaact 2820 ctgggctacg ctattggggg cacctacctg gggccagcat tccgcggact gaagggcagg 2880 accctctatc cggcagtaag cgctgtctgg ggccagtgcc aggtccgcat ccgctacctg 2940 ggcgaaagga gaggtgaggc ctggggcaga cgtggggaga actttctgtc cctggtggca 3000 gtggtttggg atggaaactc ttctgacaag agcagagggg atggaccttc atccagcctg 3060 cctcaacctc tgttcagtgc tgggaaaggc taggggtctt cacagctgtt atttaattta 3120 acccaacagc aatagaggtg aaacaggctt gagaaagcaa ctttctcaag ttctcttggc 3180 cagtaaatgg tgaaccttca gaatggaggg aggaactgca gggatgagag aattcaggag 3240 atatcaaccc ctgagcaaga ggtgcaaagc gttaggtact gggtttgatg tacaggtcca 3300 aaagaaggat gggcagagcc aggtacccag gctgtatacc ggattccctg ggctctaacc 3360 tgtctctgtg ccacatacct acttccttcc tcagccacac ctctggatgg agacactggg 3420 gccctgggca ccagggagga gagcagtgga ggaggcaggg ccttagggtg gggcagcagg 3480 ggaggagcct ccccaggaac tgactgggtc cagggcttgg agctgctctc tgcagttgtg 3540 tgggctgtag agtggagggc catccctcct cacctcagcc ccagctccca agcctctgga 3600 gtcaaagcct gggccagctc caccactgtc agagccacct tggcctgttg tttagagggc 3660 cttagccagc tcttcacccc cagctctgac tagggatgtg tgaaatctta tctgggaggc 3720 agaacttccg ggtatctcaa attccccttt cagccaggtg ggcacactcg aagcaggaaa 3780 gcagaaaggc atctgagtag gaccccgtag tttgaggaca tctggctggt ggctgcaccc 3840 atacttacat tcccctcctt ctctctccca gcggagccac actcccttct gcacctgagc 3900 cgcctgtgtg tgcgccacaa cctgggggat acccggctcg gccaggtgtc tgccctgccc 3960 ttgccccctg ccatgaagcg ctacctgctc taccagtgag ccctgtgata ccacagactg 4020 tgctgaggtc ttgccaccac ccctcccctt ggggaggtgg ggaggcactg ctggcctaga 4080 ccagctgctg aaagctggtg aggctgagcc cctaccccaa cccaagctct gcggaaatca 4140 acagccccag agccacttgg agggaggaag aaagggagcc ggcgttcaag gctatgacag 4200 tctgctacgc aaaacatttt ttcaagtaaa aatagtaaga gatgttgtta tagaaacctg 4260 ttcttgtttt tttttttttc ttgcacaaat gatcatttat atagctgcct caaaaaggaa 4320 gattatctgg gcaagtccag tgaaggcaga caaaccacaa gacctagtgc caggtttatt 4380 ccctcacatg ggtggttcac atacacagca cagaggcacg ggcaccatgg gagagggcag 4440 cactcctgcc ttctgagggg atcttggcct cacggtgtaa gaagggagag gatggtttct 4500 cttctgccct cactagggcc tagggaaccc aggagcaaat cccaccacgc cttccatctc 4560 tcagccaagg agaagccacc ttggtgacgt ttagttccaa ccattatagt aagtggagaa 4620 gggattggcc tggtcccaac cattacaggg tgaagatata aacagtaaag gaagatacag 4680 tttggatgag gccacaggaa ggagcagatg acaccatcag aagcatatgc agggaaaggg 4740 cagttactgg gcttctgggc tgcttagtcc ctggcttggc aggaagggta gggaagatgg 4800 atggggctca ttgtttggca ttgatgatgt ccacgaattc gggcttgagg gaagcaccac 4860 ccacaaggaa gccatccaca tcaggctggc tggccagctc cttgcaggtt gccccagtca 4920 cagagcctgg gaagggagca gaacaagggc ttggtcaaga atgggatgag tctgccccat 4980 ccccacctcc atgtccgagg gctcagtcta gtcctcagcc cactccacct cagccgggaa 5040 ccaaagccac tcacctccat aaatgatacg ggtgctctga gccaccgcat cagagacgtt 5100 ggacttcagc catcctcgga gcttctcgtg tacttcctgg gcctagaaca agaagctggc 5160 ctaagtaaga ccttttctgc ctctctaaga ggaaaaatca ctggcaccag tggacactta 5220 gtgtggtttc tgactgagtc agagtaccag ggctctgatc caagccaggc cctggactgg 5280 atgcccttgg acaagtcact gtctctgggt tcaaggtctc tgtgtctttg aaataagggg 5340 ttgccccatg tgggctgtgt ctgtccaaac ctattgaggc aggctgggat gagggcaggg 5400 ctcctgggcc cggttacctg ttggggtgtt gcagtcttgc cagtaccaat ggcccacaca 5460 ggctcatagg ccaggacgac cttgctccag tccttcacgt tatctgcagg gcagagatac 5520 agatggaggg aagggtgaac aagaaagagc tctccagcca ggttctccgg agtacgaaga 5580 acggtggcct actgccccct agtggacatt ggggg 5615 <210> SEQ ID NO 48 <211> LENGTH: 263 <212> TYPE: PRT <213> ORGANISM: Human <400> SEQUENCE: 48 Met Gly Gln Thr Ala Leu Ala Gly Gly Ser Ser Ser Thr Pro Thr Pro 1 5 10 15 Gln Ala Leu Tyr Pro Asp Leu Ser Cys Pro Glu Gly Leu Glu Glu Leu 20 25 30 Leu Ser Ala Pro Pro Pro Asp Leu Gly Ala Gln Arg Arg His Gly Trp 35 40 45 Asn Pro Lys Asp Cys Ser Glu Asn Ile Glu Val Lys Glu Gly Gly Leu 50 55 60 Tyr Phe Glu Arg Arg Pro Val Ala Gln Ser Thr Asp Gly Ala Arg Gly 65 70 75 80 Lys Arg Gly Tyr Ser Arg Gly Leu His Ala Trp Glu Ile Ser Trp Pro 85 90 95 Leu Glu Gln Arg Gly Thr His Ala Val Val Gly Val Ala Thr Ala Leu 100 105 110 Ala Pro Leu Gln Thr Asp His Tyr Ala Ala Leu Leu Gly Ser Asn Ser 115 120 125 Glu Ser Trp Gly Trp Asp Ile Gly Arg Gly Lys Leu Tyr His Gln Ser 130 135 140 Lys Gly Pro Gly Ala Pro Gln Tyr Pro Ala Gly Thr Gln Gly Glu Gln 145 150 155 160 Leu Glu Val Pro Glu Arg Leu Leu Val Val Leu Asp Met Glu Glu Gly 165 170 175 Thr Leu Gly Tyr Ala Ile Gly Gly Thr Tyr Leu Gly Pro Ala Phe Arg 180 185 190 Gly Leu Lys Gly Arg Thr Leu Tyr Pro Ala Val Ser Ala Val Trp Gly 195 200 205 Gln Cys Gln Val Arg Ile Arg Tyr Leu Gly Glu Arg Arg Ala Glu Pro 210 215 220 His Ser Leu Leu His Leu Ser Arg Leu Cys Val Arg His Asn Leu Gly 225 230 235 240 Asp Thr Arg Leu Gly Gln Val Ser Ala Leu Pro Leu Pro Pro Ala Met 245 250 255 Lys Arg Tyr Leu Leu Tyr Gln 260 <210> SEQ ID NO 49 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic <400> SEQUENCE: 49 agctagatct ggaccctaca atggcagc 28 <210> SEQ ID NO 50 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic <400> SEQUENCE: 50 agctagatct gccatcctac tcgaggggcc agctgg 36 <210> SEQ ID NO 51 <211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:N-terminal GFP tag <400> SEQUENCE: 51 Met Ala Arg Gln Ser Lys Gly Glu Glu Leu Phe Thr Glu Leu Tyr Lys 5 10 15 Thr Arg <210> SEQ ID NO 52 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:3′ genomic oligonucleotide no.3243 <400> SEQUENCE: 52 aagtccgttc aagtagcgca tgcggatctc 30 <210> SEQ ID NO 53 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:5′ genomic oligonucleotide no.3244 <400> SEQUENCE: 53 gagatccgca tgcgctactt gaacggactt 30 <210> SEQ ID NO 54 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Amino acid sequence encoded by SEQ ID NO:53 <400> SEQUENCE: 54 Glu Ile Arg Met Arg Tyr Leu Asn Gly Leu 5 10 <210> SEQ ID NO 55 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:3′ cDNA oligonucleotide no.3245 <400> SEQUENCE: 55 agctacgcgt ctggtagagg aggtaggctt tgag 34 <210> SEQ ID NO 56 <211> LENGTH: 822 <212> TYPE: DNA <213> ORGANISM: Mouse <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(822) <400> SEQUENCE: 56 atg ggt cag aag gtc acg gga ggg atc aag act gtg gac atg cgg gac 48 Met Gly Gln Lys Val Thr Gly Gly Ile Lys Thr Val Asp Met Arg Asp 1 5 10 15 ccc aca tac cga cct ctg aag cag gaa ctc cag ggg ctg gat tac tgc 96 Pro Thr Tyr Arg Pro Leu Lys Gln Glu Leu Gln Gly Leu Asp Tyr Cys 20 25 30 aag ccc acc cgg ctg gac ctg ctg ctc gac atg ccc ccc gtg tcc tac 144 Lys Pro Thr Arg Leu Asp Leu Leu Leu Asp Met Pro Pro Val Ser Tyr 35 40 45 gat gtg cag ctg ctc cac tcc tgg aac aat aac gac cgt tcg ctc aac 192 Asp Val Gln Leu Leu His Ser Trp Asn Asn Asn Asp Arg Ser Leu Asn 50 55 60 gtc ttc gtg aag gaa gat gac aag ttg atc ttt cac cgg cat ccg gtg 240 Val Phe Val Lys Glu Asp Asp Lys Leu Ile Phe His Arg His Pro Val 65 70 75 80 gcc cag agc acg gac gcc atc agg ggc aaa gtt ggg tac aca cgt gga 288 Ala Gln Ser Thr Asp Ala Ile Arg Gly Lys Val Gly Tyr Thr Arg Gly 85 90 95 ctg cac gta tgg cag atc aca tgg gcc atg agg cag cga ggc acg cat 336 Leu His Val Trp Gln Ile Thr Trp Ala Met Arg Gln Arg Gly Thr His 100 105 110 gcc gtg gtg ggg gtg gcc aca gca gat gcc cct ttg cac tcc gtt ggg 384 Ala Val Val Gly Val Ala Thr Ala Asp Ala Pro Leu His Ser Val Gly 115 120 125 gac aca acc ctt gta gga aat aac cat gaa tcc tgg ggc tgg gac ctg 432 Tyr Thr Thr Leu Val Gly Asn Asn His Glu Ser Trp Gly Trp Asp Leu 130 135 140 ggg cgt aac cgt ctc tac cac gac ggc aag aac cag cca agt aaa aca 480 Gly Arg Asn Arg Leu Tyr His Asp Gly Lys Asn Gln Pro Ser Lys Thr 145 150 155 160 tac cca gcc ttt ctg gag ccg gac gag aca ttc att gtc cct gac tcc 528 Tyr Pro Ala Phe Leu Glu Pro Asp Glu Thr Phe Ile Val Pro Asp Ser 165 170 175 ttt ctc gtg gcc ctg gac atg gat gat ggg acc tta agt ttc atc gtg 576 Phe Leu Val Ala Leu Asp Met Asp Asp Gly Thr Leu Ser Phe Ile Val 180 185 190 gat gga cag tac atg gga gtg gct ttc cgg gga ctc aag ggt aaa aag 624 Asp Gly Gln Tyr Met Gly Val Ala Phe Arg Gly Leu Lys Gly Lys Lys 195 200 205 ctg tat cct gta gtg agt gcc gtc tgg ggc cac tgt gag atc cgc atg 672 Leu Tyr Pro Val Val Ser Ala Val Trp Gly His Cys Glu Ile Arg Met 210 215 220 cgc tac ttg aac gga ctt gat cct gag ccc ctg cca ctc atg gac ctg 720 Arg Tyr Leu Asn Gly Leu Asp Pro Glu Pro Leu Pro Leu Met Asp Leu 225 230 235 240 tgc cgg cgt tcg gtg cgc cta gcg ctg gga aag gag cgc ctg ggt gcc 768 Cys Arg Arg Ser Val Arg Leu Ala Leu Gly Lys Glu Arg Leu Gly Ala 245 250 255 atc ccc gct ctg ccg cta cct gcc tcc ctc aaa gcc tac ctc ctc tac 816 Ile Pro Ala Leu Pro Leu Pro Ala Ser Leu Lys Ala Tyr Leu Leu Tyr 260 265 270 cag tga 822 Gln <210> SEQ ID NO 57 <211> LENGTH: 273 <212> TYPE: PRT <213> ORGANISM: Mouse <400> SEQUENCE: 57 Met Gly Gln Lys Val Thr Gly Gly Ile Lys Thr Val Asp Met Arg Asp 1 5 10 15 Pro Thr Tyr Arg Pro Leu Lys Gln Glu Leu Gln Gly Leu Asp Tyr Cys 20 25 30 Lys Pro Thr Arg Leu Asp Leu Leu Leu Asp Met Pro Pro Val Ser Tyr 35 40 45 Asp Val Gln Leu Leu His Ser Trp Asn Asn Asn Asp Arg Ser Leu Asn 50 55 60 Val Phe Val Lys Glu Asp Asp Lys Leu Ile Phe His Arg His Pro Val 65 70 75 80 Ala Gln Ser Thr Asp Ala Ile Arg Gly Lys Val Gly Tyr Thr Arg Gly 85 90 95 Leu His Val Trp Gln Ile Thr Trp Ala Met Arg Gln Arg Gly Thr His 100 105 110 Ala Val Val Gly Val Ala Thr Ala Asp Ala Pro Leu His Ser Val Gly 115 120 125 Tyr Thr Thr Leu Val Gly Asn Asn His Glu Ser Trp Gly Trp Asp Leu 130 135 140 Gly Arg Asn Arg Leu Tyr His Asp Gly Lys Asn Gln Pro Ser Lys Thr 145 150 155 160 Tyr Pro Ala Phe Leu Glu Pro Asp Glu Thr Phe Ile Val Pro Asp Ser 165 170 175 Phe Leu Val Ala Leu Asp Met Asp Asp Gly Thr Leu Ser Phe Ile Val 180 185 190 Asp Gly Gln Tyr Met Gly Val Ala Phe Arg Gly Leu Lys Gly Lys Lys 195 200 205 Leu Tyr Pro Val Val Ser Ala Val Trp Gly His Cys Glu Ile Arg Met 210 215 220 Arg Tyr Leu Asn Gly Leu Asp Pro Glu Pro Leu Pro Leu Met Asp Leu 225 230 235 240 Cys Arg Arg Ser Val Arg Leu Ala Leu Gly Lys Glu Arg Leu Gly Ala 245 250 255 Ile Pro Ala Leu Pro Leu Pro Ala Ser Leu Lys Ala Tyr Leu Leu Tyr 260 265 270 Gln <210> SEQ ID NO 58 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Oligonucleotide no.3342 <400> SEQUENCE: 58 agctggcgcg ccagggtcag aaggtcacgg gaggg 35 <210> SEQ ID NO 59 <211> LENGTH: 128 <212> TYPE: PRT <213> ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY: UNSURE <222> LOCATION: (1) <223> OTHER INFORMATION: Xaa is Leu, Ile, Val, Met, Ala or Pro <221> NAME/KEY: UNSURE <222> LOCATION: (2) <223> OTHER INFORMATION: Xaa is any amino acid residue <221> NAME/KEY: UNSURE <222> LOCATION: (3) <223> OTHER INFORMATION: Xaa is Pro, Thr or Ser <221> NAME/KEY: UNSURE <222> LOCATION: (4) <223> OTHER INFORMATION: Xaa is Leu, Ile, Val, Met, Ala or Pro <221> NAME/KEY: UNSURE <222> LOCATION: (5) <223> OTHER INFORMATION: Xaa is any amino acid <221> NAME/KEY: UNSURE <222> LOCATION: (6) <223> OTHER INFORMATION: Xaa is any amino acid <221> NAME/KEY: UNSURE <222> LOCATION: (7) <223> OTHER INFORMATION: Xaa is Leu, Ile, Val, Met, Ala, Phe, Tyr or Trp <221> NAME/KEY: UNSURE <222> LOCATION: (8) <223> OTHER INFORMATION: Xaa is Cys, Thr or Ser <221> NAME/KEY: UNSURE <222> LOCATION: (9) <223> OTHER INFORMATION: Xaa is Arg, Lys or His <221> NAME/KEY: UNSURE <222> LOCATION: (10) <223> OTHER INFORMATION: Xaa is any amino acid <221> NAME/KEY: UNSURE <222> LOCATION: (11) <223> OTHER INFORMATION: Xaa is any amino acid <221> NAME/KEY: UNSURE <222> LOCATION: (12) <223> OTHER INFORMATION: Xaa is Leu, Ile, Val, Met, Ala or Pro <221> NAME/KEY: UNSURE <222> LOCATION: (13) <223> OTHER INFORMATION: Xaa is any amino acid <221> NAME/KEY: UNSURE <222> LOCATION: (14) <223> OTHER INFORMATION: Xaa is any amino acid <221> NAME/KEY: UNSURE <222> LOCATION: (15) <223> OTHER INFORMATION: Xaa is any amino acid <221> NAME/KEY: UNSURE <222> LOCATION: (16) <223> OTHER INFORMATION: Xaa is Leu, Ile, Val, Met, Ala, Pro, Gly, Cys, Thr or Ser <221> NAME/KEY: UNSURE <222> LOCATION: (17)..(66) <223> OTHER INFORMATION: Xaa can be any amino acid or no amino acid. Position 17-66 can be 1-50 amino acids. <221> NAME/KEY: UNSURE <222> LOCATION: (67) <223> OTHER INFORMATION: Xaa is Leu, Ile, Val, Met, Ala or Pro <221> NAME/KEY: UNSURE <222> LOCATION: (68) <223> OTHER INFORMATION: Xaa is any amino acid <221> NAME/KEY: UNSURE <222> LOCATION: (69) <223> OTHER INFORMATION: Xaa is any amino acid <221> NAME/KEY: UNSURE <222> LOCATION: (70) <223> OTHER INFORMATION: Xaa is Leu, Ile, Val, Met, Ala or Pro <221> NAME/KEY: UNSURE <222> LOCATION: (72) <223> OTHER INFORMATION: Xaa is Leu, Ile, Val, Met, Ala, Pro or Gly <221> NAME/KEY: UNSURE <222> LOCATION: (73) <223> OTHER INFORMATION: Xaa is Pro or Asn <221> NAME/KEY: UNSURE <222> LOCATION: (74)..(123) <223> OTHER INFORMATION: Xaa can be any amino acid or no amino acid. Position 74-123 can be 0-50 amino acids. <221> NAME/KEY: UNSURE <222> LOCATION: (124) <223> OTHER INFORMATION: Xaa is Leu, Ile, Val, Met, Ala or Pro <221> NAME/KEY: UNSURE <222> LOCATION: (125)..(128) <223> OTHER INFORMATION: Xaa is any amino acid <400> SEQUENCE: 59 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 <210> SEQ ID NO 60 <211> LENGTH: 1611 <212> TYPE: DNA <213> ORGANISM: Mouse <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1611) <400> SEQUENCE: 60 atg gat aaa gtg ggg aaa atg tgg aac aac tta aaa tac aga tgc cag 48 Met Asp Lys Val Gly Lys Met Trp Asn Asn Leu Lys Tyr Arg Cys Gln 1 5 10 15 aat ctc ttc agc cac gag gga gga agc cgt aat gag aac gtg gag atg 96 Asn Leu Phe Ser His Glu Gly Gly Ser Arg Asn Glu Asn Val Glu Met 20 25 30 aac ccc aac aga tgt ccg tct gtc aaa gag aaa agc atc agt ctg gga 144 Asn Pro Asn Arg Cys Pro Ser Val Lys Glu Lys Ser Ile Ser Leu Gly 35 40 45 gag gca gct ccc cag caa gag agc agt ccc tta aga gaa aat gtt gcc 192 Glu Ala Ala Pro Gln Gln Glu Ser Ser Pro Leu Arg Glu Asn Val Ala 50 55 60 tta cag ctg gga ctg agc cct tcc aag acc ttt tcc agg cgg aac caa 240 Leu Gln Leu Gly Leu Ser Pro Ser Lys Thr Phe Ser Arg Arg Asn Gln 65 70 75 80 aac tgt gcc gca gag atc cct caa gtg gtt gaa atc agc atc gag aaa 288 Asn Cys Ala Ala Glu Ile Pro Gln Val Val Glu Ile Ser Ile Glu Lys 85 90 95 gac agt gac tcg ggt gcc acc cca gga acg agg ctt gca cgg aga gac 336 Asp Ser Asp Ser Gly Ala Thr Pro Gly Thr Arg Leu Ala Arg Arg Asp 100 105 110 tcc tac tcg cgg cac gcc ccg tgg gga gga aag aag aaa cat tcc tgt 384 Ser Tyr Ser Arg His Ala Pro Trp Gly Gly Lys Lys Lys His Ser Cys 115 120 125 tcc aca aag acc cag agt tca ttg gat acc gag aaa aag ttt ggt aga 432 Ser Thr Lys Thr Gln Ser Ser Leu Asp Thr Glu Lys Lys Phe Gly Arg 130 135 140 act cga agc ggc ctt cag agg cga gag cgg cgc tat gga gtc agc tcc 480 Thr Arg Ser Gly Leu Gln Arg Arg Glu Arg Arg Tyr Gly Val Ser Ser 145 150 155 160 atg cag gac atg gac agc gtt tct agc cgc acg gtc ggg agc cgc tcc 528 Met Gln Asp Met Asp Ser Val Ser Ser Arg Thr Val Gly Ser Arg Ser 165 170 175 ctg agg cag agg ctc cag gac acg gtg ggt ttg tgt ttt ccc atg aga 576 Leu Arg Gln Arg Leu Gln Asp Thr Val Gly Leu Cys Phe Pro Met Arg 180 185 190 act tac agc aag cag tca aag cca ctc ttt tcc aat aaa aga aaa ata 624 Thr Tyr Ser Lys Gln Ser Lys Pro Leu Phe Ser Asn Lys Arg Lys Ile 195 200 205 cat ctt tct gaa tta atg ctg gag aaa tgc cct ttt cct gct ggc tcg 672 His Leu Ser Glu Leu Met Leu Glu Lys Cys Pro Phe Pro Ala Gly Ser 210 215 220 gat tta gca caa aag tgg cat ttg att aaa cag cat acc gcc cct gtg 720 Asp Leu Ala Gln Lys Trp His Leu Ile Lys Gln His Thr Ala Pro Val 225 230 235 240 agc cca cac tca aca ttt ttt gat aca ttt gat cca tca ctg gtg tct 768 Ser Pro His Ser Thr Phe Phe Asp Thr Phe Asp Pro Ser Leu Val Ser 245 250 255 aca gaa gat gaa gaa gat agg ctt cgc gag aga aga cgg ctt agt atc 816 Thr Glu Asp Glu Glu Asp Arg Leu Arg Glu Arg Arg Arg Leu Ser Ile 260 265 270 gaa gaa ggg gtg gat ccc cct ccc aac gca caa ata cac acc ttt gaa 864 Glu Glu Gly Val Asp Pro Pro Pro Asn Ala Gln Ile His Thr Phe Glu 275 280 285 gct act gca cag gtc aac cca ttg tat aag ctg gga cca aag tta gct 912 Ala Thr Ala Gln Val Asn Pro Leu Tyr Lys Leu Gly Pro Lys Leu Ala 290 295 300 cct ggg atg aca gag ata agt gga gat ggt tct gca att cca caa acg 960 Pro Gly Met Thr Glu Ile Ser Gly Asp Gly Ser Ala Ile Pro Gln Thr 305 310 315 320 aat tgt gac tca gaa gag gat tca acc acc cta tgt ctg cag tca cgg 1008 Asn Cys Asp Ser Glu Glu Asp Ser Thr Thr Leu Cys Leu Gln Ser Arg 325 330 335 agg cag aag cag cgc cag gtg tcc ggg gac agc cac gcg cac gtt agc 1056 Arg Gln Lys Gln Arg Gln Val Ser Gly Asp Ser His Ala His Val Ser 340 345 350 aga cag gga gct tgg aaa gtt cat acg cag atc gat tac ata cac tgc 1104 Arg Gln Gly Ala Trp Lys Val His Thr Gln Ile Asp Tyr Ile His Cys 355 360 365 ctc gtg cca gat ttg ctt cag atc aca ggg aat ccc tgt tac tgg ggc 1152 Leu Val Pro Asp Leu Leu Gln Ile Thr Gly Asn Pro Cys Tyr Trp Gly 370 375 380 gtg atg gac cga tac gag gcc gaa gcc ctt cta gaa ggg aaa ccg gaa 1200 Val Met Asp Arg Tyr Glu Ala Glu Ala Leu Leu Glu Gly Lys Pro Glu 385 390 395 400 ggc acg ttc ttg ctc agg gac tct gca cag gag gac tac ctc ttc tct 1248 Gly Thr Phe Leu Leu Arg Asp Ser Ala Gln Glu Asp Tyr Leu Phe Ser 405 410 415 gtg agc ttc cgc cgc tac aac agg tct ctg cac gcc cgg atc gag cag 1296 Val Ser Phe Arg Arg Tyr Asn Arg Ser Leu His Ala Arg Ile Glu Gln 420 425 430 tgg aac cac aac ttc agc ttc gat gcc cat gac ccc tgc gtg ttt cac 1344 Trp Asn His Asn Phe Ser Phe Asp Ala His Asp Pro Cys Val Phe His 435 440 445 tcc tcc aca gtc acg ggg ctt ctc gaa cac tat aaa gac ccc agc tct 1392 Ser Ser Thr Val Thr Gly Leu Leu Glu His Tyr Lys Asp Pro Ser Ser 450 455 460 tgc atg ttt ttt gaa ccg ttg cta acg ata tca ctg aat aga act ttc 1440 Cys Met Phe Phe Glu Pro Leu Leu Thr Ile Ser Leu Asn Arg Thr Phe 465 470 475 480 cct ttc agc ctg cag tat atc tgc cgc gca gtg atc tgc aga tgc act 1488 Pro Phe Ser Leu Gln Tyr Ile Cys Arg Ala Val Ile Cys Arg Cys Thr 485 490 495 acg tat gat ggg att gac ggg ctc ccg cta ccg tcg atg tta cag gat 1536 Thr Tyr Asp Gly Ile Asp Gly Leu Pro Leu Pro Ser Met Leu Gln Asp 500 505 510 ttt tta aaa gag tat cat tat aaa caa aaa gtt agg gtt cgc tgg tta 1584 Phe Leu Lys Glu Tyr His Tyr Lys Gln Lys Val Arg Val Arg Trp Leu 515 520 525 gaa cga gag cca gtc aaa gca aag taa 1611 Glu Arg Glu Pro Val Lys Ala Lys 530 535 <210> SEQ ID NO 61 <211> LENGTH: 536 <212> TYPE: PRT <213> ORGANISM: Mouse <400> SEQUENCE: 61 Met Asp Lys Val Gly Lys Met Trp Asn Asn Leu Lys Tyr Arg Cys Gln 1 5 10 15 Asn Leu Phe Ser His Glu Gly Gly Ser Arg Asn Glu Asn Val Glu Met 20 25 30 Asn Pro Asn Arg Cys Pro Ser Val Lys Glu Lys Ser Ile Ser Leu Gly 35 40 45 Glu Ala Ala Pro Gln Gln Glu Ser Ser Pro Leu Arg Glu Asn Val Ala 50 55 60 Leu Gln Leu Gly Leu Ser Pro Ser Lys Thr Phe Ser Arg Arg Asn Gln 65 70 75 80 Asn Cys Ala Ala Glu Ile Pro Gln Val Val Glu Ile Ser Ile Glu Lys 85 90 95 Asp Ser Asp Ser Gly Ala Thr Pro Gly Thr Arg Leu Ala Arg Arg Asp 100 105 110 Ser Tyr Ser Arg His Ala Pro Trp Gly Gly Lys Lys Lys His Ser Cys 115 120 125 Ser Thr Lys Thr Gln Ser Ser Leu Asp Thr Glu Lys Lys Phe Gly Arg 130 135 140 Thr Arg Ser Gly Leu Gln Arg Arg Glu Arg Arg Tyr Gly Val Ser Ser 145 150 155 160 Met Gln Asp Met Asp Ser Val Ser Ser Arg Thr Val Gly Ser Arg Ser 165 170 175 Leu Arg Gln Arg Leu Gln Asp Thr Val Gly Leu Cys Phe Pro Met Arg 180 185 190 Thr Tyr Ser Lys Gln Ser Lys Pro Leu Phe Ser Asn Lys Arg Lys Ile 195 200 205 His Leu Ser Glu Leu Met Leu Glu Lys Cys Pro Phe Pro Ala Gly Ser 210 215 220 Asp Leu Ala Gln Lys Trp His Leu Ile Lys Gln His Thr Ala Pro Val 225 230 235 240 Ser Pro His Ser Thr Phe Phe Asp Thr Phe Asp Pro Ser Leu Val Ser 245 250 255 Thr Glu Asp Glu Glu Asp Arg Leu Arg Glu Arg Arg Arg Leu Ser Ile 260 265 270 Glu Glu Gly Val Asp Pro Pro Pro Asn Ala Gln Ile His Thr Phe Glu 275 280 285 Ala Thr Ala Gln Val Asn Pro Leu Tyr Lys Leu Gly Pro Lys Leu Ala 290 295 300 Pro Gly Met Thr Glu Ile Ser Gly Asp Gly Ser Ala Ile Pro Gln Thr 305 310 315 320 Asn Cys Asp Ser Glu Glu Asp Ser Thr Thr Leu Cys Leu Gln Ser Arg 325 330 335 Arg Gln Lys Gln Arg Gln Val Ser Gly Asp Ser His Ala His Val Ser 340 345 350 Arg Gln Gly Ala Trp Lys Val His Thr Gln Ile Asp Tyr Ile His Cys 355 360 365 Leu Val Pro Asp Leu Leu Gln Ile Thr Gly Asn Pro Cys Tyr Trp Gly 370 375 380 Val Met Asp Arg Tyr Glu Ala Glu Ala Leu Leu Glu Gly Lys Pro Glu 385 390 395 400 Gly Thr Phe Leu Leu Arg Asp Ser Ala Gln Glu Asp Tyr Leu Phe Ser 405 410 415 Val Ser Phe Arg Arg Tyr Asn Arg Ser Leu His Ala Arg Ile Glu Gln 420 425 430 Trp Asn His Asn Phe Ser Phe Asp Ala His Asp Pro Cys Val Phe His 435 440 445 Ser Ser Thr Val Thr Gly Leu Leu Glu His Tyr Lys Asp Pro Ser Ser 450 455 460 Cys Met Phe Phe Glu Pro Leu Leu Thr Ile Ser Leu Asn Arg Thr Phe 465 470 475 480 Pro Phe Ser Leu Gln Tyr Ile Cys Arg Ala Val Ile Cys Arg Cys Thr 485 490 495 Thr Tyr Asp Gly Ile Asp Gly Leu Pro Leu Pro Ser Met Leu Gln Asp 500 505 510 Phe Leu Lys Glu Tyr His Tyr Lys Gln Lys Val Arg Val Arg Trp Leu 515 520 525 Glu Arg Glu Pro Val Lys Ala Lys 530 535 <210> SEQ ID NO 62 <211> LENGTH: 1601 <212> TYPE: DNA <213> ORGANISM: Mouse <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1599) <400> SEQUENCE: 62 atg aag aaa atc agt ctg aag acc ttc agg aaa tct ttt aac ctg agt 48 Met Lys Lys Ile Ser Leu Lys Thr Phe Arg Lys Ser Phe Asn Leu Ser 1 5 10 15 aaa agc aaa gac gaa act gag ttc atg gtg gtt cag ccc cag tcc ctt 96 Lys Ser Lys Asp Glu Thr Glu Phe Met Val Val Gln Pro Gln Ser Leu 20 25 30 gct ggt gac ttc gtg aaa gat gac tct tta ttc ggg agc tgt tat ggc 144 Ala Gly Asp Phe Val Lys Asp Asp Ser Leu Phe Gly Ser Cys Tyr Gly 35 40 45 aaa gac atg gcc agt tgt gac att ggc agc gag gat gag aaa ggg aag 192 Lys Asp Met Ala Ser Cys Asp Ile Gly Ser Glu Asp Glu Lys Gly Lys 50 55 60 aac aga tcc aaa agc gag agc ctg atg ggc act ttg aag agg cgg ttg 240 Asn Arg Ser Lys Ser Glu Ser Leu Met Gly Thr Leu Lys Arg Arg Leu 65 70 75 80 tcc gcc aag cag aag acc aag ggc aag ggc ggc act gcg tct aca gat 288 Ser Ala Lys Gln Lys Thr Lys Gly Lys Gly Gly Thr Ala Ser Thr Asp 85 90 95 gag gac acc ttc tcc tca gct tca gct cct ggt ggg ctc aag gat gtg 336 Glu Asp Thr Phe Ser Ser Ala Ser Ala Pro Gly Gly Leu Lys Asp Val 100 105 110 cgt gct ccg cgg ccc atc cgc tcc aca tca ctg aga agc cac cat tat 384 Arg Ala Pro Arg Pro Ile Arg Ser Thr Ser Leu Arg Ser His His Tyr 115 120 125 agc ccc acg ccc tgg ccg ctg cgt ccc acc agc tcg gag gag acg tgc 432 Ser Pro Thr Pro Trp Pro Leu Arg Pro Thr Ser Ser Glu Glu Thr Cys 130 135 140 atc aag atg gag atg cga gtg aaa gca ctg gtg cat gct gcc agc cca 480 Ile Lys Met Glu Met Arg Val Lys Ala Leu Val His Ala Ala Ser Pro 145 150 155 160 gga cca gtc aac ggt gtg cgc aag gat ctg cgg gag cta cag ccc agg 528 Gly Pro Val Asn Gly Val Arg Lys Asp Leu Arg Glu Leu Gln Pro Arg 165 170 175 gag ctg cga gac ctg cag cca gag ccg cgc cct gag tcc cgc tgc agc 576 Glu Leu Arg Asp Leu Gln Pro Glu Pro Arg Pro Glu Ser Arg Cys Ser 180 185 190 ccc agc tca ccc ggg gac ctg agc ctc cac ctg gag gaa cac gtg cct 624 Pro Ser Ser Pro Gly Asp Leu Ser Leu His Leu Glu Glu His Val Pro 195 200 205 gta gta atc gga ctc atg tct cag gac tac ctt cag tac acc gtg cct 672 Val Val Ile Gly Leu Met Ser Gln Asp Tyr Leu Gln Tyr Thr Val Pro 210 215 220 tta gat gac ggg atg tgc cct ctt gaa ggg ccg cgc agc tgc tgc ctg 720 Leu Asp Asp Gly Met Cys Pro Leu Glu Gly Pro Arg Ser Cys Cys Leu 225 230 235 240 gat acg tct tct ccc atg gag gtg tca gcc gta ccc ctg ccg ggg gcg 768 Asp Thr Ser Ser Pro Met Glu Val Ser Ala Val Pro Leu Pro Gly Ala 245 250 255 agt ggt gcc ttc tcc gaa gac gac agt cat gtg gac cag gac ctg gtt 816 Ser Gly Ala Phe Ser Glu Asp Asp Ser His Val Asp Gln Asp Leu Val 260 265 270 gta ggc cca gag atc ctt gtg gat tca tca gtg aac aat ttg ttg att 864 Val Gly Pro Glu Ile Leu Val Asp Ser Ser Val Asn Asn Leu Leu Ile 275 280 285 ggc acc aca gga gtc atg ttg cag agc cct aga gga ggt cat gat gac 912 Gly Thr Thr Gly Val Met Leu Gln Ser Pro Arg Gly Gly His Asp Asp 290 295 300 gcc cct ccc ctc tca cca ttg cta cct cca atg cag aat aac cca atc 960 Ala Pro Pro Leu Ser Pro Leu Leu Pro Pro Met Gln Asn Asn Pro Ile 305 310 315 320 caa agg aac ttc agt ggc ctc tcg ggc cca gac ttg cac atg gcc gaa 1008 Gln Arg Asn Phe Ser Gly Leu Ser Gly Pro Asp Leu His Met Ala Glu 325 330 335 agt gtt cgc tgt cat ttg aat ttc gat ccc aac tct gcg cct ggg gtt 1056 Ser Val Arg Cys His Leu Asn Phe Asp Pro Asn Ser Ala Pro Gly Val 340 345 350 gct aga gtt tat gac tcg gtg caa agt agt ggc ccc atg gtt gtt aca 1104 Ala Arg Val Tyr Asp Ser Val Gln Ser Ser Gly Pro Met Val Val Thr 355 360 365 agt ctt acg gag gag ctg aag aag ctt gca aaa cag ggg tgg tat tgg 1152 Ser Leu Thr Glu Glu Leu Lys Lys Leu Ala Lys Gln Gly Trp Tyr Trp 370 375 380 ggc ccc atc aca cgc tgg gag gca gag ggg aag ttg gca aat gtg cca 1200 Gly Pro Ile Thr Arg Trp Glu Ala Glu Gly Lys Leu Ala Asn Val Pro 385 390 395 400 gat ggt tct ttt ctt gta agg gat agt tct gat gac cgt tac ctt tta 1248 Asp Gly Ser Phe Leu Val Arg Asp Ser Ser Asp Asp Arg Tyr Leu Leu 405 410 415 agc ctg agc ttt cgt tcc cat ggt aaa aca ctt cac act aga att gag 1296 Ser Leu Ser Phe Arg Ser His Gly Lys Thr Leu His Thr Arg Ile Glu 420 425 430 cac tca aat ggt aga ttc agc ttt tat gaa cag cca gat gtg gaa ggg 1344 His Ser Asn Gly Arg Phe Ser Phe Tyr Glu Gln Pro Asp Val Glu Gly 435 440 445 cat aca tct ata gtt gac tta atc gag cat tca atc agg gac tct gaa 1392 His Thr Ser Ile Val Asp Leu Ile Glu His Ser Ile Arg Asp Ser Glu 450 455 460 aat gga gca ttt tgt tat tca aga tct cga ttg cct gga tca gca act 1440 Asn Gly Ala Phe Cys Tyr Ser Arg Ser Arg Leu Pro Gly Ser Ala Thr 465 470 475 480 tac cca gtc aga ctg acc aat cca gtg tca cga ttc atg cag gtg cgc 1488 Tyr Pro Val Arg Leu Thr Asn Pro Val Ser Arg Phe Met Gln Val Arg 485 490 495 tcg ctg cag tac ctg tgc cgc ttt gtt atc cgt cag tac acc aga ata 1536 Ser Leu Gln Tyr Leu Cys Arg Phe Val Ile Arg Gln Tyr Thr Arg Ile 500 505 510 gac tta att cag aaa ctg cct ttg cca aac aaa atg aag gat tat ttg 1584 Asp Leu Ile Gln Lys Leu Pro Leu Pro Asn Lys Met Lys Asp Tyr Leu 515 520 525 cag gag aag cac tac tg 1601 Gln Glu Lys His Tyr 530 <210> SEQ ID NO 63 <211> LENGTH: 533 <212> TYPE: PRT <213> ORGANISM: Mouse <400> SEQUENCE: 63 Met Lys Lys Ile Ser Leu Lys Thr Phe Arg Lys Ser Phe Asn Leu Ser 1 5 10 15 Lys Ser Lys Asp Glu Thr Glu Phe Met Val Val Gln Pro Gln Ser Leu 20 25 30 Ala Gly Asp Phe Val Lys Asp Asp Ser Leu Phe Gly Ser Cys Tyr Gly 35 40 45 Lys Asp Met Ala Ser Cys Asp Ile Gly Ser Glu Asp Glu Lys Gly Lys 50 55 60 Asn Arg Ser Lys Ser Glu Ser Leu Met Gly Thr Leu Lys Arg Arg Leu 65 70 75 80 Ser Ala Lys Gln Lys Thr Lys Gly Lys Gly Gly Thr Ala Ser Thr Asp 85 90 95 Glu Asp Thr Phe Ser Ser Ala Ser Ala Pro Gly Gly Leu Lys Asp Val 100 105 110 Arg Ala Pro Arg Pro Ile Arg Ser Thr Ser Leu Arg Ser His His Tyr 115 120 125 Ser Pro Thr Pro Trp Pro Leu Arg Pro Thr Ser Ser Glu Glu Thr Cys 130 135 140 Ile Lys Met Glu Met Arg Val Lys Ala Leu Val His Ala Ala Ser Pro 145 150 155 160 Gly Pro Val Asn Gly Val Arg Lys Asp Leu Arg Glu Leu Gln Pro Arg 165 170 175 Glu Leu Arg Asp Leu Gln Pro Glu Pro Arg Pro Glu Ser Arg Cys Ser 180 185 190 Pro Ser Ser Pro Gly Asp Leu Ser Leu His Leu Glu Glu His Val Pro 195 200 205 Val Val Ile Gly Leu Met Ser Gln Asp Tyr Leu Gln Tyr Thr Val Pro 210 215 220 Leu Asp Asp Gly Met Cys Pro Leu Glu Gly Pro Arg Ser Cys Cys Leu 225 230 235 240 Asp Thr Ser Ser Pro Met Glu Val Ser Ala Val Pro Leu Pro Gly Ala 245 250 255 Ser Gly Ala Phe Ser Glu Asp Asp Ser His Val Asp Gln Asp Leu Val 260 265 270 Val Gly Pro Glu Ile Leu Val Asp Ser Ser Val Asn Asn Leu Leu Ile 275 280 285 Gly Thr Thr Gly Val Met Leu Gln Ser Pro Arg Gly Gly His Asp Asp 290 295 300 Ala Pro Pro Leu Ser Pro Leu Leu Pro Pro Met Gln Asn Asn Pro Ile 305 310 315 320 Gln Arg Asn Phe Ser Gly Leu Ser Gly Pro Asp Leu His Met Ala Glu 325 330 335 Ser Val Arg Cys His Leu Asn Phe Asp Pro Asn Ser Ala Pro Gly Val 340 345 350 Ala Arg Val Tyr Asp Ser Val Gln Ser Ser Gly Pro Met Val Val Thr 355 360 365 Ser Leu Thr Glu Glu Leu Lys Lys Leu Ala Lys Gln Gly Trp Tyr Trp 370 375 380 Gly Pro Ile Thr Arg Trp Glu Ala Glu Gly Lys Leu Ala Asn Val Pro 385 390 395 400 Asp Gly Ser Phe Leu Val Arg Asp Ser Ser Asp Asp Arg Tyr Leu Leu 405 410 415 Ser Leu Ser Phe Arg Ser His Gly Lys Thr Leu His Thr Arg Ile Glu 420 425 430 His Ser Asn Gly Arg Phe Ser Phe Tyr Glu Gln Pro Asp Val Glu Gly 435 440 445 His Thr Ser Ile Val Asp Leu Ile Glu His Ser Ile Arg Asp Ser Glu 450 455 460 Asn Gly Ala Phe Cys Tyr Ser Arg Ser Arg Leu Pro Gly Ser Ala Thr 465 470 475 480 Tyr Pro Val Arg Leu Thr Asn Pro Val Ser Arg Phe Met Gln Val Arg 485 490 495 Ser Leu Gln Tyr Leu Cys Arg Phe Val Ile Arg Gln Tyr Thr Arg Ile 500 505 510 Asp Leu Ile Gln Lys Leu Pro Leu Pro Asn Lys Met Lys Asp Tyr Leu 515 520 525 Gln Glu Lys His Tyr 530 <210> SEQ ID NO 64 <211> LENGTH: 34 <212> TYPE: PRT <213> ORGANISM: Mus musculus or Rattus norvegicus <400> SEQUENCE: 64 Val Arg Pro Leu Gln Glu Leu Cys Arg Gln Arg Ile Val Ala Ala Val 1 5 10 15 Gly Arg Glu Asn Leu Ala Arg Ile Pro Leu Asn Pro Val Leu Arg Asp 20 25 30 Tyr Leu <210> SEQ ID NO 65 <211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 65 Ala Pro Thr Leu Gln His Phe Cys Arg Leu Ala Ile Asn Lys Cys Thr 1 5 10 15 Gly Thr Ile Trp Gly Leu Pro Leu Pro Thr Arg Leu Lys Asp Tyr Leu 20 25 30 <210> SEQ ID NO 66 <211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 66 Val Ala Thr Leu Gln His Leu Cys Arg Lys Thr Val Asn Gly His Leu 1 5 10 15 Asp Ser Tyr Glu Lys Val Thr Gln Leu Pro Gly Pro Ile Arg Glu Phe 20 25 30 Leu <210> SEQ ID NO 67 <211> LENGTH: 34 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 67 Val Arg Pro Leu Gln Glu Leu Cys Arg Gln Arg Ile Val Ala Thr Val 1 5 10 15 Gly Arg Glu Asn Leu Ala Arg Ile Pro Leu Asn Pro Val Leu Arg Asp 20 25 30 Tyr Leu <210> SEQ ID NO 68 <211> LENGTH: 34 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 68 Val Pro Ser Leu Gln His Ile Cys Arg Met Ser Ile Arg Arg Val Met 1 5 10 15 Ser Thr Gln Glu Val Gln Lys Leu Pro Val Pro Ser Lys Ile Leu Ala 20 25 30 Phe Leu <210> SEQ ID NO 69 <211> LENGTH: 34 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 69 Pro Phe Ser Leu Gln Tyr Ile Cys Arg Ala Val Ile Cys Arg Cys Thr 1 5 10 15 Thr Tyr Asp Gly Ile Asp Gly Leu Pro Leu Pro Ser Met Leu Gln Asp 20 25 30 Phe Leu <210> SEQ ID NO 70 <211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 70 Pro Arg Thr Leu Leu Ser Leu Cys Arg Val Ala Val Arg Arg Ala Leu 1 5 10 15 Gly Lys Tyr Arg Leu His Leu Val Pro Ser Leu Pro Leu Pro Asp Pro 20 25 30 Ile Lys Lys Phe Leu 35 <210> SEQ ID NO 71 <211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 71 Pro Arg Ser Leu Gln His Leu Cys Arg Cys Ala Leu Arg Ser His Leu 1 5 10 15 Glu Gly Cys Leu Pro His Ala Leu Pro Arg Leu Pro Leu Pro Pro Arg 20 25 30 Met Leu Arg Phe Leu 35 <210> SEQ ID NO 72 <211> LENGTH: 34 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 72 Val Arg Ser Leu Gln Tyr Leu Cys Arg Phe Val Ile Cys Gln Tyr Thr 1 5 10 15 Arg Ile Asp Leu Ile Gln Lys Leu Pro Leu Pro Asn Lys Met Lys Asp 20 25 30 Tyr Leu <210> SEQ ID NO 73 <211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 73 Pro Arg Pro Leu Ala His Leu Cys Arg Leu Arg Val Arg Lys Ala Ile 1 5 10 15 Gly Lys Tyr Arg Ile Lys Leu Leu Asp Thr Leu Pro Leu Pro Gly Arg 20 25 30 Leu Ile Arg Tyr Leu 35 <210> SEQ ID NO 74 <211> LENGTH: 34 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 74 Val Lys Ser Leu Gln His Leu Cys Arg Phe Arg Ile Arg Gln Tyr Thr 1 5 10 15 Arg Ile Asp His Ile Pro Asp Leu Pro Leu Pro Lys Pro Leu Ile Ser 20 25 30 Tyr Ile <210> SEQ ID NO 75 <211> LENGTH: 40 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 75 Val Pro Ser Leu Thr His Leu Cys Arg Leu Glu Ile Arg Ala Ser Leu 1 5 10 15 Lys Ala Glu His Leu His Ser Asp Ile Phe Ile His Gln Leu Pro Leu 20 25 30 Pro Arg Ser Leu Gln Asn Tyr Leu 35 40 <210> SEQ ID NO 76 <211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 76 Pro Leu Pro Leu Met Asp Leu Cys Arg Arg Ser Val Arg Leu Ala Leu 1 5 10 15 Gly Lys Glu Arg Leu Gly Ala Ile Pro Ala Leu Pro Leu Pro Ala Ser 20 25 30 Leu Lys Ala Tyr Leu 35 <210> SEQ ID NO 77 <211> LENGTH: 34 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 77 Pro Phe Ser Leu Gln His Ile Cys Arg Thr Val Ile Cys Asn Cys Thr 1 5 10 15 Thr Tyr Asp Gly Ile Asp Ala Leu Pro Ile Pro Ser Pro Met Lys Leu 20 25 30 Tyr Leu <210> SEQ ID NO 78 <211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 78 Pro Gln Ser Leu Leu His Leu Ser Arg Leu Cys Val Arg His Ala Leu 1 5 10 15 Gly Asp Thr Arg Leu Gly Gln Ile Ser Thr Leu Pro Leu Pro Pro Ala 20 25 30 Met Lys Arg Tyr Leu 35 <210> SEQ ID NO 79 <211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 79 Pro His Ser Leu Leu His Leu Ser Arg Leu Cys Val Arg His Asn Leu 1 5 10 15 Gly Asp Thr Arg Leu Gly Gln Val Ser Ala Leu Pro Leu Pro Pro Ala 20 25 30 Met Lys Arg Tyr Leu 35 <210> SEQ ID NO 80 <211> LENGTH: 34 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 80 Leu Ser Ser Leu Lys His Leu Cys Arg Lys Ala Leu Arg Ser Phe Leu 1 5 10 15 Thr Thr Tyr Gln Val Leu Ala Leu Pro Ile Pro Lys Lys Met Lys Glu 20 25 30 Phe Leu <210> SEQ ID NO 81 <211> LENGTH: 34 <212> TYPE: PRT <213> ORGANISM: Mouse <400> SEQUENCE: 81 Val Arg Ser Leu Gln Tyr Leu Cys Arg Phe Val Ile Arg Gln Tyr Thr 1 5 10 15 Arg Ile Asp Leu Ile Gln Lys Leu Pro Leu Pro Asn Lys Met Lys Asp 20 25 30 Tyr Leu 

What is claimed is:
 1. An isolated protein comprising a SOCS box, wherein the SOCS box comprises the amino acid sequence: X₁X₂X₃X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆[X_(i)]_(n) X₁₇ X₁₈ X₁₉ X₂₀ X₂₁ X₂₂ X₂₃ [X_(j)]_(n) X₂₄ X₂₆ X₂₇ X₂₈ (SEQ ID NO: 59) wherein: X₁ is L, I, V, M, A or P; X₂ is any amino acid residue; X₃ is P, T or S; X₄ is L, I, V, M, A or P; X₅ is any amino acid; X₆ is any amino acid; X₇ is L, I, V, M, A, F, Y or W; X₈ is C, T or S; X₉ is R, K or H; X₁₀ is any amino acid; X₁₁ is any amino acid; X₁₂ is L, I, V, M, A or P; X₁₃ is any amino acid; X₁₄ is any amino acid; X₁₅ is any amino acid; X₁₆ is L, I, V, M, A, P. G, C, T or S; [X_(i)]_(n) is a sequence of n amino acids wherein n is from 1 to 50 amino acids and wherein the sequence X₁ can comprises the same or different amino acids selected from any amino acid residue; X₁₇ is L, I, V, M, A or P; X₁₈ is any amino acid; X₁₉ is any amino acid; X₂₀ L, I, V, M, A or P; X₂₁ is P; X₂₂ is L, I, V, M, A, P or G; X₂₃ is P or N; [X_(j)]_(n) is a sequence of n amino acids wherein n is from 0 to 50 amino acids and wherein the sequence X_(j) can comprise the same or different amino acids selected from any amino acid residue; X₂₄ is L, I, V, M, A or P; X₂₅ is any amino acid; X₂₆ is any amino acid; X₂₇ is Y or F; and X₂₈ is L, I, V, M, A or P; with the proviso that said protein is not CIS.
 2. An isolated protein comprising a SOCS box, wherein the SOCS box comprises the amino acid sequence: X₁ X₂ X₃ X₄ X₅ X₆ X_(7 l X) ₈ X₉ X₁₀ X₁₁ X₁₂ X₁₃ X₁₄ X₁₅ X₁₆ [X_(i)]_(n) X₁₇ X₁₈ X₁₉ X₂₀ X₂₁ X₂₂ X₂₃ [Xj]_(n) X₂₄ X₂₅ X₂₆ X₂₇ X₂₈ (SEQ ID NO:59) wherein: X₁ is L, I, V, M or P; X₂ is any amino acid residue; X₃ is P, T or S; X₄ is L, I, V, M, A or P; X₅ is any amino acid; X₆ is any amino acid; X₇ is L, I, V, M, A, F, Y or W; X₈ is C, T or S; X₉ is R, K or H; X₁₀ is any amino acid; X₁₁ is any amino acid; X₁₂ is L, I, V, M, A or P; X₁₃ is any amino acid; X₁₄ is any amino acid; X₁₅ is any amino acid; X₁₆ is L, I, V, M, A, P, G, C, T or S; [X_(i)]_(n) is a sequence of n amino acids wherein n is from 1 to 50 amino acids and wherein the sequence X_(i) can comprise the same or different amino acids selected from any amino acid residue; X₁₇ is L, I, V, M, A or P; X₁₈ is any amino acid; X₁₉ is any amino acid; X₂₀ L, I, V, M, A or P; X₂₁ is P; X₂₂ is L, I, V, M, A, P or G; X₂₃ is P or N; [X_(j)]_(n) is a sequence of n amino acids wherein n is from 0 to 50 amino acids and wherein the sequence X_(j) can comprise the same of different amino acids selected from any amino acid residue; X₂₄ is L, I, V, M, A or P; X₂₅ is any amino acid; X₂₆ is any amino acid; X₂₇ is Y or F; and X₂₈ is L, I, V, M, A or P.
 3. An isolated protein comprising a SOCS box, wherein said SOCS box comprises a sequence selected from any one of SEQ ID NOs: 64-81.
 4. An isolated protein comprising a SOCS box, wherein said SOCS box comprises a sequence having at least about 70% similarity to any one of SEQ ID NOs: 64-81.
 5. The isolated protein according to anyone of claim 1 or 2-4 wherein said protein modulates signal transduction.
 6. The isolated protein according to claim 5 wherein said signal transduction is modulated by an effector molecule selected from a cytokine, a hormone, a microbe or a microbial product, a parasite, or an antigen.
 7. The isolated protein according to claim 6 wherein said protein modulates cytokine-mediated signal transduction.
 8. An isolated protein according to claim 7 wherein the signal transduction is mediated by at least one of the cytokines EPO, TPO, G-CSF, GM-CSF, IL-3, IL-2, IL-4, IL-7, IL-13, IL-6, LIF, IL-12, ILFNγ, TNF, IL-1 or M-CSF.
 9. An isolated protein according to claim 8 wherein the signal transduction is mediated by at least one of IL-6, LIF, OSM, IFM-γ or thrombopoieten.
 10. An isolated protein according to claim 9 wherein the signal transduction is mediated by IL-6.
 11. The isolated protein according to anyone of claims 1 or 2, wherein said protein comprises an amino acid sequence as set forth in any one of [<400>] SEQ ID NOS: 4, [<400>] 6, [<400>] 8, [<400>] 10, [<400>]12, [<400>] 14, [<400>] 18, [<400>] 21, [<400>] 25, [<400>] 29, [<400>] 36, [<400>] 41, [<400>] 44, [<400>] 46, [<400>] 48, 57, 61, or 63, or an amino acid sequence having at least about 50% similarity to any one of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 18, 21, 25, 29, 36, 41, 44, 46, 48, 57, 61 or
 63. 12. The isolated protein according to any one claim 1 or 2, wherein the said protein is encoded by a nucleotide sequence as set forth in any one of [<400>] SEQ ID NOS: 3, [<400>]5, [<400>]7, [<400>]9, [<400>]11, [<400>]13, [<400>] 15, [<400>] 16, [<400>] 17, [<400>] 20, [<400>] 22, [<400>] 23, [<400>] 24, [<400>] 26, [<400>] 27, [<400>] 28, [<400>] 30, [<400>] 31, [<400>] 32, [<400>] 33, [<400>] 34, [<400>] 35, [<400>] 37, [<400>] 38, [<400>] 39, [<400>] 40, [<400>] 42, [<400>] 43, [<400>] 45, [or <400>] 47, 56, 60, or 62, or a nucleotide sequence having at least about 50% identity to any one of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15-17, 20, 22-24, 26-28, 30-35, 37-40, 42-43, 45, 47, 56, 60 or 62; or a nucleotide sequence which hybridises to any one of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15-17, 20, 22-24, 26-28, 30-35, 37-40, 42-43, 45, 47, 56, 60 or 62 under low stringency conditions, wherein said low stringency conditions comprise at least about 1% v/v to about 15%v/v formamide and at least about 1M to about 2M salt for hybridisation at 42° C., and at least about 1M to about 2M salt for washing.
 13. The isolated protein according to anyone of claim 1 or 2, further comprising a domain in a region N-terminal of said SOCS box, wherein said domain is an SH2 domain, a domain comprising WD-40 repeats, or a domain comprising ankyrin repeats.
 14. The isolated protein of claim 7 wherein said protein modulates cytokine-mediated signal transduction by inhibiting the activity of JAK kinases.
 15. The isolated protein of claim 7 wherein said protein modulates cytokine-mediated signal transduction by reducing the phosphorylation of cell receptors and STATs. 